Cell culture medium

ABSTRACT

The invention relates to serum-free cell culture medium, wherein the medium either contains maltose as the sole carbohydrate source, or contains maltose and at least one other saccharide as carbohydrate sources. In a preferred embodiment, the additional saccharide is a monosaccharide, preferably glucose, and the medium comprises DMEM-F12. In a preferred embodiment the cells to be cultured may be CHO or HEK293 cells. Also disclosed are methods of growing and/or culturing a cell using the cell culture medium as described herein, methods of increasing protein yield using the cell culture medium as described herein, and kits thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of Singapore patentapplication No. 10201601284Y, filed 22 Feb. 2016, the contents of itbeing hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to compositions for propagation,preservation, or maintenance of cells. In particular, the presentinvention relates to culture media. The present invention also relatesto compositions for supporting protein production by cultured cells.

BACKGROUND OF THE INVENTION

From the first day of its inception, cell culture medium hasprogressively been improved and researched depending on the intention ofthe culture. The first few cell culture solution provides irrigation,supply of water, inorganic anions required for cell metabolism, osmoticbalance, a buffering system, and a carbohydrate source for growth.

As research into how cells function improves, cell culture medium hasevolved to include multitude of components, which may be includeddepending on the purpose of the medium. However, one that remains aconstant for cells (such as mammalian cells) are the provision ofglucose as a carbohydrate source.

As such, there is a need to provide cell culture medium that providesalternative carbohydrate source. The potential use of alternativecarbohydrate source, as energy source can have practical implications inbiopharmaceutical manufacturing. In particular, the potential use ofalternative carbohydrate source may lead to the improvement ofcarbohydrate loading in the batch culture medium, and possibly lead tothe decrease lactate accumulation which may become toxic to cells.

Accordingly, there is a need to provide a cell culture medium that hasan alternative carbohydrate source.

SUMMARY OF THE INVENTION

In first aspect, there is provided a serum-free cell culture medium. Thecell culture medium comprises maltose as sole carbohydrate source.

In second aspect, there is provided a serum-free cell culture mediumcomprising maltose and at least one, at least two, at least three ormore saccharides as carbohydrate sources.

In third aspect, there is provided a method of growing and/or culturinga cell, wherein the method comprises growing and/or culturing a cell inthe serum-free cell culture medium of the first aspect or the secondaspect.

In fourth aspect, there is provided a method of growing and/or culturinga cell, wherein the method comprising growing and/or culturing a cell inthe serum-free cell culture medium as described herein.

In fifth aspect, there is provided a method of increasing protein yield,wherein the method comprises growing and/or culturing a cell in theserum-free cell culture medium as described herein.

In sixth aspect, there is provided a kit comprising the components ofthe serum-free cell culture medium as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the accompanying drawings, in which:

FIG. 1 shows the results of adaptability tests of Chinese Hamster Ovary(CHO) cells in various disaccharide media. In (A) CHO-K1 cells werecultivated in a serum-free protein-free cell culture medium, HyQ PF-CHO,with 3.6 g/l of different sugars (maltose, sucrose, lactose, trehaloseor glucose) as carbohydrate source. The viable cell densities (linedmarkers) and culture viabilities (marker only) of these cultures at thebeginning and end of each passage over a period of 74 days were plotted.Cultures with sucrose, lactose or trehalose were terminated on Day 31due to the decreased culture viabilities and reduced viable celldensities. The experiment was repeated with a seeding cell density of1.0×10⁶ cells/ml to obtain similar results. In (B) CHO-DG44 cells werecultivated with a seeding cell density of 1.0×10⁶ cells/ml in aserum-free protein-free cell culture medium, HyQ PF-CHO, with 10 g/l ofdifferent sugars (maltose, sucrose, lactose, trehalose or glucose) ascarbohydrate source. The viable cell densities (lined markers) and cellviabilities (marker only) of these cultures at the beginning and end ofeach passage over a period of 22 days were plotted. In (C) HEK293 cellswere cultivated with a seeding cell density of 1.0×10⁶ cells/ml in aprotein-free chemically defined cell culture medium, PFCDM, with 10 g/lof different sugars (maltose, sucrose, lactose, trehalose or glucose) ascarbohydrate source. The viable cell densities (lined markers) and cellviabilities (marker only) of these cultures at the beginning and end ofeach passage over a period of 22 days were plotted. Cultures withsucrose, lactose or trehalose were terminated on Day 14 due to thedecreased culture viabilities and reduced viable cell densities.Therefore, FIG. 1 demonstrates that CHO-K1, CHO-DG44 and HEK293 cellscan proliferate in serum-free culture medium utilizing maltose, but notsucrose, lactose or trehalose, as sugar source.

FIG. 2 shows growth and biochemical profiles of CHO-K1 cells adapted toand cultivated in maltose medium, compared to non-adapted CHO-K1 cellscultivated in glucose medium. CHO-K1 cells that were pre-adapted to aserum-free protein-free cell culture medium, HyQ PF-CHO with 3.6 g/l ofmaltose as carbohydrate source (square marker) and normal non-adaptedCHO-K1 cells cultivated in the same culture medium but with glucose asthe carbohydrate source (cross) were monitored over 14 days, to obtaintheir (A) Viable cell densities (lined marker) and culture viabilities(marker only), and (B) Glucose, (C) Lactate, (D) Glutamine and (E)Ammonium profiles. To illustrate the specific consumption and productionrates of (F) Glutamine and (G) Ammonium, the concentrations of thesecomponents were also plotted against integral viable cell density(IVCD). The averages and standard deviations from three (3) replicateshake flasks were plotted. Thus, FIG. 2 shows that the maltose can beused as a glucose-replacement.

FIG. 3 shows growth and biochemical profiles of a recombinant monoclonalantibody producing CHO-K1 cell line (SH87) cultivated in protein-freechemically defined medium (PFCDM) with both glucose and maltose as sugarsource. SH87 cell routinely maintained in glucose-only PFCDM wassub-cultivated into PFCDM with 4 g/l glucose, 4 g/l glucose+0.5 g/lmaltose, 4 g/l glucose+1 g/l maltose, 4 g/l glucose+3 g/l maltose, or 6g/l glucose. The cultures were monitored till culture viabilities werelower than 50%, to obtain their (A) Viable cell densities (lined marker)and culture viabilities (marker only), and (B) Glucose, (C) Lactate, (D)Maltose, (E) IgG titer, (F) Glutamine, and (G) Ammonium concentrationsin the culture media. (H) SH87 cells sub-cultivated in PFCDM with 4 g/lglucose, or 4 g/l glucose+3 g/l maltose, were harvested from Days 0 to 5for LC-MS quantification of intracellular maltose concentrations.Intracellular maltose was found to be absent (below detection limit) inall samples obtained from the 4 g/l glucose cultures and in the samplefrom Day 0 of the 4 g/l glucose+3 g/l maltose cultures. The averages andstandard deviations of two (2) technical replicates from one set ofshake flasks were plotted for (D). For the other profiles, the averagesand standard deviations from two (2) replicate shake flasks wereplotted. Thus, FIG. 3 shows that cells can switch to maltose metabolismupon glucose depletion, to sustain cell growth and protein production.

FIG. 4 shows maltose and glucose profiles in cell-free conditioned media(CM). In (A) SH87 cell were routinely maintained in glucose-only PFCDMwas sub-cultivated into PFCDM with 4 g/l glucose, or 4 g/l glucose+3 g/lmaltose. On Days 2, 4, 6 and 8, cell-free conditioned medium (CM) wereharvested from both cultures. Viable cell densities (lined marker) andculture viabilities (marker only) were also monitored. In (B) CM fromthe maltose supplemented culture were incubated for a further 3 days at37° C. Samples were harvested daily from these CM, starting from the dayof harvest, to determine glucose and maltose concentrations. In (C) CMharvested from the glucose culture were spiked with maltose to a finalconcentration of 3 g/l prior to further incubation at 37° C. Samplesfrom these CM were harvested daily for a further three (3) days todetermine glucose and maltose concentrations. The averages and standarddeviations from two (2) replicate shake flasks were plotted. Thus, FIG.4 shows that cells cultured utilises maltose and that maltose hydrolysiswas not occurring spontaneously in the conditioned culture media, evenwhen culture viabilities were low.

FIG. 5 shows growth and biochemical profiles of SH87 cultivated inprotein-free chemically defined medium (PFCDM) with high concentrationsof glucose and maltose. SH87 cell routinely maintained in glucose-onlyPFCDM was sub-cultivated into PFCDM with 14 g/l glucose, 24 g/l glucose,4 g/l glucose+10 g/l maltose, or 4 g/l glucose+20 g/l maltose. Thecultures were monitored till culture viabilities were lower than 50%, toobtain their (A) Viable cell densities (lined marker) and cultureviabilities (marker only), and (B) Glucose, (C) Lactate, (D) Maltose,(E) Glutamine, (F) Osmolality, and (G) IgG titer profiles. For themaltose profile, the averages and standard deviations of two (2)technical replicates from one set of shake flasks were plotted. For theother profiles, the averages and standard deviations from two (2)replicate shake flasks were plotted. Thus, FIG. 5 shows similar cellgrowth in cultures with 10 g/l maltose supplement or 20 g/l maltosesupplement, as compared with 14 g/l glucose, thus shows that maltosemetabolism rate at higher maltose supplement concentration providesample support to cell growth. This allows for lower initial glucoseconcentrations in the cultures, leading to lactate consumption in thecell cultures with maltose supplement, as compared to the culture with14 g/l glucose. Additionally, osmolality is much lower than the culturewith 20 g/l glucose, which removes the osmolality limitation oncarbohydrate loading in cell culture media. Additionally, FIG. 5 alsodemonstrates an improvement in maximum recombinant protein production(i.e. IgG titer) in maltose supplemented culture.

FIG. 6 shows Monod model for specific maltose consumption rate. In (A),specific maltose consumption rates for cultures with different initialmaltose concentrations from different experiments were determined as theslope from the plots of maltose concentrations against cumulativeintegral viable cell densities (IVCD), according to Equation 4. In (B),maximum specific maltose consumption rate (qs_max) and affinity constant(Ks) were determined by non-linear regression, according to Equation 5.The Monod model for specific maltose consumption rate calculated usingthese parameters and the experimental data used for the model were thenplotted. Thus, FIG. 6 shows a new estimate of mammalian cell maltosemetabolism kinetics.

FIG. 7 shows growth and biochemical profiles of SH87 fed-batchbioreactor cultures in glucose only or glucose+maltose protein-freechemically defined medium (PFCDM) base medium. SH87 cell routinelymaintained in glucose-only PFCDM was sub-cultivated into PFCDM with 4g/l glucose or 4 g/l glucose+20 g/l maltose in 2 liter stirred tankbioreactors. The cultures with glucose only base medium were fed dailywith 100% of its calculated glucose requirement while the cultures withglucose and maltose base medium was fed daily with 50% of its calculatedglucose requirement. Other nutrients were fed similarly in a separatefeed using glutamine as reference nutrient. The cultures were monitoredtill culture viabilities were lower than 50%, to obtain their (A) Viablecell densities (lined marker) and culture viabilities (marker only), and(B) Glucose, (C) Lactate, (D) Maltose, (E) Glutamine, (F) Osmolality,and (G) IgG titer profiles. Duplicate bioreactor cultures were performedfor each fed-batch conditions and data from all 4 cultures are plotted.Thus, FIG. 7 shows that the presence of maltose enables the use of alower glucose feed that can result in higher maximum recombinant proteinproduction (i.e. IgG titers).

FIG. 8 shows glycosylation profiles of anti-Her2 antibody produced fromSH87 fedbatch bioreactor cultures in glucose only or glucose+maltoseprotein-free chemically defined medium (PFCDM) base medium. Anti-Her2monoclonal antibodies were purified from samples from Day 10 and Day 15of the SH87 fedbatch cultures in PFCDM with 4 g/l glucose or 4 g/lglucose+20 g/l maltose (FIG. 7), and subjected to glycosylationprofiling. (A) are representative fluorescence chromatograms of the fourDay 10 samples with symbolic representations of selected structures. (B)shows relative abundance of glycan structures. Average and standarddeviation of the two (2) biological replicates were shown. The averagedifference between Glucose+Maltose and Glucose only samples, as well asthat between Day 15 and Day 10 samples were also calculated. Effects ofmaltose supplementation and late Day 15 harvest on the relativeabundance of glycan structures were bold and underlined. p-values werecalculated using paired one-tail Student's t-Test on data fromindividual samples. Thus, FIG. 8 shows that maltose supplementation canbe used as a means to fine-tune protein co-translational orpost-translational modification processes (such as in monoclonalantibody glycosylation profile, especially in marginally reducing thesialylation level, which is known to improve antibody-dependent cellularcytotoxicity (ADCC) of therapeutic antibodies).

FIG. 9 shows growth and biochemical profiles of SH87 fed-batchbioreactor cultures in glucose+maltose protein-free chemically definedmedium (PFCDM) base medium with a glucose-only feed or a glucose+maltosefeed. SH87 cell routinely maintained in glucose-only PFCDM wassub-cultivated into PFCDM with 4 g/l glucose+20 g/l maltose in 2 Lstirred tank bioreactors. The cultures were fed daily with 50% of itscalculated glucose requirement, using a sugar feed consisting of onlyglucose, or of glucose and maltose in a 1:1 ratio. Other nutrients werefed similarly in a separate feed using glutamine as reference nutrient.The cultures were monitored till culture viabilities were lower than50%, to obtain their (A) Viable cell densities (lined marker) andculture viabilities (marker only), and (B) Glucose, (C) Lactate, (D)Maltose, (E) Glutamine, (F) Osmolality, and (G) IgG titer profiles.Duplicate bioreactor cultures were performed for each fed-batchconditions, and the averages and standard deviations from replicatecultures were plotted. Thus, FIG. 9 shows maltose supplementation in thefeed medium can decreased the lactate concentration marginally whencompared to the culture with glucose only feed. This feature will beadvantageous in cultures whereby lactate build-up is causing the cultureto crash. FIG. 9 also shows that the addition of maltose in the feedmedium can maintain a higher maltose concentration in the culture withno adverse effect on cell growth and recombinant protein production.

FIG. 10 shows growth and biochemical profiles of CHO-K1 cells in proteinfree chemically defined medium (PFCDM) with increasing concentration ofmaltose. CHO-K1 cells adapted to PFCDM with 10 g/L of maltose wascultivated in PFCDM with 10, 20, 30 and 40 g/L maltose in batch shakeflask cultures. The cultures were monitored till culture viabilitieswere lower than 50%, to obtain their (A) Viable cell densities (linedmarker) and culture viabilities (marker only), and (B) Osmolality, (C)Glucose, (D) Lactate, (E) Glutamine and (F) Ammonium profiles. Thus,FIG. 10 shows that the CHO-K1 cells can grow faster in PFCDM with 20 g/lor more maltose as the only carbohydrate in the medium. Even with thehigher maltose concentration, lactate concentration was kept at a lowlevel till viability started to drop on Day 6. This supports thefindings described in FIG. 2, which demonstrates that maltose can beused as a glucose-replacement in the absence of serum and hydrolysatesin routine cultivation of cells (such as mammalian cells), with theadditional advantage of low lactate accumulation.

FIG. 11 shows specific IgG productivities of SH87 cultivated inprotein-free chemically defined medium (PFCDM) with high concentrationsof glucose and maltose. SH87 cell routinely maintained in glucose-onlyPFCDM was sub-cultivated into PFCDM with 14 g/l glucose, 24 g/l glucose,4 g/l glucose+10 g/l maltose, or 4 g/l glucose+20 g/l maltose. IgGtiters were plotted against IVCD, according to Equation 3. The slope ofthe graphs gave the specific IgG productivities of the cultures over thetime periods. Thus, FIG. 11 supports the data in FIG. 5, where maltosecan, in some examples, improve protein productions (i.e. IgGproductivities) after glutamine depletion.

FIG. 12 shows specific maltose consumption rates of SH87 cultivated inprotein-free chemically defined medium (PFCDM). SH87 cell routinelymaintained in glucose-only PFCDM was sub-cultivated into PFCDMcontaining 4 g/l glucose and supplemented with 0.5, 1, 2, 3, 10 and 20g/l maltose. Maltose concentrations were plotted against IVCD, accordingto Equation 4. The slope of the graphs gave the specific maltoseconsumption rates of the cultures over the time periods. The 0.5, 1 and3 g/l maltose cultures were described in FIG. 3; The 10 g/l Maltose (1)and 20 g/l Maltose (1) cultures were described in FIG. 5; Detailedculture data for 2 g/l Maltose, 10 g/l Maltose (2) and 20 g/l Maltose(2) was not described in this report. Thus, FIG. 12 supports the data inFIG. 6, that demonstrates that specific maltose consumption rates may bemodelled using Monod equation.

FIG. 13 shows growth and biochemical profiles of SH87 fed-batchbioreactor cultures in glucose only protein-free chemically definedmedium (PFCDM) base medium with glucose fed at 100% or 50% of calculatedglucose requirements. SH87 cell routinely maintained in glucose-onlyPFCDM was sub-cultivated into PFCDM with 4 g/l glucose in 2 litrestirred tank bioreactors. The cultures were fed daily with either 100%of its calculated glucose or with 50% of its calculated glucoserequirement. Other nutrients were fed similarly in a separate feed usingglutamine as reference nutrient. The cultures were monitored tillculture viabilities were lower than 50%, to obtain their (A) Viable celldensities (lined marker) and culture viabilities (marker only), and (B)Glucose, (C) Lactate, (D) IgG titer, (E) Glutamine, and (F) Ammoniumprofiles. Duplicate bioreactor cultures were performed for eachfed-batch condition, and the averages and standard deviations from thereplicate bioreactor cultures were plotted. Osmolality maintained below381 mOsm/kg (data not shown). Thus, FIG. 13 shows that the use of alower glucose feed alone without maltose supplementation results in alower cell growth profile and recombinant protein production (i.e. IgGtiter). This supports the results in FIG. 7, and confirms that thepresence of maltose enabled the use of a lower glucose feed that canresult in the observed higher cell production yield (i.e. higher maximumIgG titers).

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Most cultured cells (such as mammalian cells) are chemoheterotrophic andtypically require a carbohydrate source for growth in cultures. Ascarbohydrates have low permeability through the phospholipid bilayerthat makes the bulk of the cell membrane (Bresseleers et al., 1984; Woodet al., 1968), sugar transport into the cell is facilitated bytransporter proteins (Jones and Nickson, 1982; Thorens, 1996; Wright etal., 1994). Hence, for the cultivation of cells (such as mammaliancells), glucose is the single most commonly used carbohydrate, becauseit can be efficiently transported into the cells through two majorfamilies of monosaccharide transporters, the sodium-glucose linkedtransporters (SGLT) (Wright et al., 1994) and glucose transporters(GLUT).

In addition to glucose, other carbohydrate sources have been tested fortheir ability to support growth of animal cell cultures (Altamirano etal., 2000; Morgan and Faik, 1981). In these reports, monosaccharidesgalactose, fructose and mannose were demonstrated to be utilized by mostcell types in both serum and serum-free culture media, consistent withthe availability of transporter proteins to internalize these sugars(Mueckler and Thorens, 2013; Wright, 2013). Polysaccharides had alsobeen shown to support cell growth in cell cultures supplemented withserum, because serum contains saccharidases that were essential for thebreaking down of the complex carbohydrates in the culture media (Morganand Faik, 1981). In another study, heat inactivated serum devoid ofamylase and/or maltase activities and culture dishes coated withserum-containing medium were used to isolate Chinese Hamster Ovary (CHO)cell variants that can utilize maltose or starch (Scannell and Morgan,1982). The authors showed that the culture dish coated withserum-containing medium did not contribute to saccharidase activity, andthus they hypothesized that endogenous carbohydrate hydrolases,otherwise only expressed in the small intestines, were induced in theseisolates to allow for their growth in maltose and starch-containingmedia (Scannell and Morgan, 1982). Nonetheless, saccharidase-containingserum was used in this study to coat the culture dishes, and how thiscontributed to cell utilization of maltose and starch was not evaluated.

In this study, the use of disaccharides to support the growth of amammalian Chinese Hamster Ovary (CHO) cell line was evaluated, CHO-K1,in a serum-free protein-free culture. CHO-K1 cells was found to becapable of utilizing maltose for growth in the absence of glucose. Usinga production CHO-K1 cell line producing a recombinant monoclonalantibody, these cells were shown to be capable of utilizing maltoseafter glucose depletion in a biphasic manner, when culture media withboth glucose and maltose were used. In addition, it was demonstratedthat maltose was internalized by the cells and did not hydrolyzespontaneously in the conditioned culture media. Maltose supplementationalso led to a 15% improvement in the cell protein production (i.e.recombinant monoclonal antibody titer from batch culture). The specificmaltose consumption rates were then determined and fitted in a Monodmodel to obtain a maximum specific maltose consumption rate (qs_max) of0.257 ng/cell/day and an affinity constant (Ks) of 7.03 g/l. Theapplication of maltose supplementation in fed-batch bioreactor cultureswere then demonstrated to result in 23% and 55% improvements in maximummonoclonal antibody titers and specific monoclonal antibodyproductivities respectively, when compared to glucose-only fed-batchcultures. Hence, maltose supplementation may be applied as a simplebioreactor process modification to improve cell protein production (suchas monoclonal antibody) yields in current manufacturing processes.

The inventors of the present disclosure surprisingly found that there isno report to-date on the use of polysaccharides to support cell growthin serum-free cultivation of cells (such as mammalian cells), eventhough serum-free and protein free cultivation of mammalian cells hasbeen reported since the 1970s and 1980s respectively (Hayashi and Sato,1976; Okabe et al., 1984). This is not surprising, since there is onlyone known animal disaccharide sucrose transporter that was recentlyreported (Meyer et al., 2011). Whether polysaccharides can supportmammalian cell growth in serum-free culture is of interest for bothbasic and applied sciences. For the basic understanding of mammaliancell metabolism of polysaccharides, the use of serum-free culture cancompletely preclude the role of saccharidase from serum contributing tothe survival of cells utilizing only polysaccharides, which could not beruled out in the previous report (Scannell and Morgan, 1982). If a serumfree mammalian cell culture utilizing polysaccharides is obtained, theculture can be a model to elucidate yet unknown mechanisms ofpolysaccharide transport and metabolism in mammalian cells, such as therecent discovery of the first known animal sucrose transporter inDrosophila melanogaster (Meyer et al., 2011).

For practical applications, the use of polysaccharides for mammaliancell cultivation can give advantages to the serum free suspension cellculture of transformed cell lines typically used or the biomanufacturingof recombinant protein therapeutics. Two glucose-related issues arecommonly encountered for such cell cultures: Firstly, glucose iscommonly the limiting substrate in serum-free suspension cell batchculture due its high consumption rate and the high cell density attainedin suspension cell cultures. This is despite the high initial glucoseconcentration in the culture media, as glucose is commonly the mostabundant nutrient in most media formulations for mammalian cell batchculture (Sinacore et al., 2000), at 2 to 10 fold higher concentrationthan the next most abundant nutrient utilized by the cells during cellgrowth. Further loading of the cell culture media with glucose islimited by the overall osmolality of the culture media sincehyperosmotic culture media has been shown to be detrimental to cellgrowth (Kurano et al., 1990; Ozturk and Palsson, 1991). Secondly,glucose is known to contribute to high lactate levels in culture, sincethese transformed cell lines have high rates of glycolysis and lactateproduction, a phenotype described as the Warburg effect (Warburg, 1956).This becomes a productivity limitation to both batch and fed-batchcultures, because lactate is toxic to the cells and increased lactateconcentrations in the bioreactor will result in decreased cell growthrate (Hassell et al., 1991; Lao and Toth, 1997).

The inventors of the present disclosure, thus, hypothesised that the useof polysaccharides in serum free suspension cell culture may potentiallyaddress both issues: As polysaccharides contribute to lower osmolalityper unit mass concentration, these sugars can potentially increase thesugar availability to cells in batch culture media since higher massconcentrations can be used. Depending on the rate of conversion of thepolysaccharides to monosaccharides, it may also mitigate lactateaccumulation in the bioreactor by providing a source of sugars that isnot readily available to the cells, thereby limiting glycolysis andlactate production. In theory, this will be somewhat similar tomaintaining low glucose concentrations in bioreactors as achieved bydynamic online feeding strategies (Wong et al., 2005), albeit beingpractically simpler to setup and implement.

In view of the above, in the present disclosure, the use ofdisaccharides, the simplest polysaccharides, to support the growth of amammalian cell line in a serum-free protein-free culture is described.Therefore, in a first aspect, the present invention refers to aserum-free cell culture medium comprising maltose as sole carbohydratesource. In another aspect, the present invention refers to serum-freecell culture medium comprising maltose and at least one additional, atleast two additional, at least three additional or more additionalsaccharides as carbohydrate sources.

As illustrated in the Experimental Section below, the inventors of thepresent disclosure surprisingly demonstrated the successful use ofmaltose as a carbohydrate source in mammalian cell cultivation. It issurprisingly shown that animal cells are able to utilize disaccharides.The result was surprising because mammalian cells are not known toexpress disaccharide transporters, and only selected mammalian cells(such as intestinal cells) expresses maltases on its cell membrane toallow the digestion of maltose.

As used herein, the term “serum-free” refers to a cell culture mediumthat is devoid of fetal calf serum (FCS) or fetal bovine serum (FBS) ornew-born calf serum (NBS), or serum from any other human or animalorigin. As illustrated or demonstrated in the Experimental Sectionbelow, the inventors of the present inventors were able to use maltoseas a carbohydrate source in serum free cultures. The use of maltose ascarbohydrate source in the present disclosure is in contrast with theuses known in the art where maltose are generally used as a carbohydratesource for cell (mammalian cell) cultivation in serum containing media.The reason for the use of maltose for cell (mammalian cell) cultivationin serum containing media is because serum has been shown to containmaltases, which will break down maltose to glucose in vitro prior tometabolism by the cells. The inventors of the present disclosuredeveloped the surprising use of disaccharides as a source ofcarbohydrate for serum-free cell culture (such as animal cell culture).Furthermore, it is also surprisingly shown that cells (such as mammaliancells) can be routinely cultivated in maltose containing media in theabsence of glucose, as they can in glucose containing media.

The advantage of using maltose in serum free cell culture (such asmammalian cell culture) is that it can be added to cell culture mediawith less osmolality load, since it is a disaccharide. Hence, morecarbohydrate can be loaded to the culture media compared to amonosaccharide such as glucose. In addition, as demonstrated herewith,the use of maltose in a batch culture medium can result in loweredlactate accumulation, which is also known to be detrimental to cellcultivation. It has also been demonstrated in the present disclosurethat the use of maltose in batch and fed-batch culture can improveprotein (such as recombinant protein or recombinant monoclonal antibody)production.

In some examples, the cell culture as described herein may providecarbohydrate source that may solely be maltose. In some examples, thecell culture as described herein may provide carbohydate source that mayhave at least one additional (i.e. maltose plus at least one more), orat least two, or at least three, or at least four, or at least five, orat least six, or at least seven, or at least eight, or moresaccharide(s). As described in the Experimental Section below, the batchculture media were loaded with saccharides, such that carbohydrates areno longer limiting. Thus, in some examples, the saccharide may be,independently, or in combination with one another, present at aconcentration of between 0.5 g/litre to 40 g/litre, between 10 g/litreto 15 g/litre, between 15 g/litre to 20 g/litre, between 20 g/litre to25 g/litre, between 25 g/litre to 30 g/litre, between 30 g/litre to 35g/litre, between 35 g/litre to 40 g/litre, at least about 1 g/litre, atleast about 2 g/litre, at least about 3 g/litre, at least about 4g/litre, at least about 5 g/litre, at least about 8 g/litre, at leastabout 10 g/litre, at least about 15 g/litre, at least about 20 g/litre,at least about 25 g/litre, at least about 30 g/litre, at least about 35g/litre, about 1.5 g/litre, about 3.5 g/litre, about 3.6 g/litre, about5.5 g/litre, about 8 g/litre, about 11 g/litre, about 14 g/litre, about18 g/litre, about 23 g/litre, about 28 g/litre, about 33 g/litre, orabout 38 g/litre.

In some examples, the saccharide as described herein may be apolysaccharide. The polysaccharide as described herein may be a glucanor a disaccharide.

As used herein, the term “glucan” refers to a polysaccharide that is apolymer made up of D-glucose monomers linked together by glycosidicbonds. In some examples, the glucan as described herein may include, butis not limited to, cellobiose, kojibiose, nigerose, isomaltose,β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose,dextran, glycogen, pullulan, starch, cellulose, chrysolaminarin,curdlan, laminarin, lentinan, lichenin, oat beta-glucan, pleuran,zymosan, and combinations thereof.

In some examples, the “disaccharide” as described herein may include,but is not limited to, cellobiose, chitobiose, kojibiose, nigerose,isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose,gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose,melibiose, melibiulose, rutinose, rutinulose, xylobiose and combinationsthereof.

In some examples, the saccharide as described herein may be componentsof undefined cell culture supplements including, but is not limited to,soy hydrolysates, yeastolates, lactalbumin hydrolysates, caseinhydrolysates, gelatin hydrolysates, gluten hydrolysates, liverhydrolysates, vegetable hydrolysates, wheat hydrolysates, peptone fromanimal tissues, tryptose, protein peptones and combinations thereof.

In some examples, the saccharide as described herein may bemonosaccharide. As used herein, the term “monosaccharide” refers to anyof a class of carbohydrates that cannot be broken down to simpler sugarsby hydrolysis and that constitute the building blocks ofoligosaccharides and polysaccharides. Monosaccharides may have at leastthree carbon atoms, one of which is attached to an oxygen atom to forman aldehyde group (CHO) or a ketone, and the others of which are eachattached to a hydroxyl group (OH). A monosaccharide that comprises threecarbons per molecule is referred to a triose. A monosaccharide thatcomprises four carbons per molecule is referred to as a tetrose. Amonosaccharide that comprises five carbons per molecule is referred toas a pentose. A monosaccharide sugar containing six carbons per moleculeis referred to as a hexose. Monosaccharides can occur as chains orrings. Monosaccharides may include, but is not limited to, tagatose,glucose, galactose, ribose, fructose and xylose. As shown in theexperimental section, when maltose is used, improvement in protein titerwas observed. Thus, in some example, the monosaccharide may be glucose.As shown in the Experimental section of the present disclosure, in someexamples, the culture medium as described herein may comprise glucosewith additional maltose supplement. Thus, in some example, the cellculture medium as described herein may comprise maltose and glucose assole carbohydrate sources. As demonstrated in the Experimental Sectionbelow, using maltose in cell culture (such as fed-batch processes)allows the control of glucose at lower levels to improve recombinantprotein production. Without wishing to be bound by theory, thecombination of maltose and glucose as sole carbohydrate sources mayreduce the risk of complete carbohydrate depletion that can cause theculture to die off (crash).

As described in the Experimental Section below, the batch culture mediawere loaded with maltose, such that carbohydrates are no longerlimiting. Thus, in some examples, the maltose may be present in theculture medium as described herein at a concentration of between 0.5g/litre to 40 g/litre, between 10 g/litre to 15 g/litre, between 15g/litre to 20 g/litre, between 20 g/litre to 25 g/litre, between 25g/litre to 30 g/litre, between 30 g/litre to 35 g/litre, between 35g/litre to 40 g/litre, at least about 1 g/litre, at least about 2g/litre, at least about 3 g/litre, at least about 4 g/litre, at leastabout 5 g/litre, at least about 8 g/litre, at least about 10 g/litre, atleast about 15 g/litre, at least about 20 g/litre, at least about 25g/litre, at least about 30 g/litre, at least about 35 g/litre, about 1.5g/litre, about 3.5 g/litre, about 3.6 g/litre, about 5.5 g/litre, about8 g/litre, about 11 g/litre, about 14 g/litre, about 18 g/litre, about23 g/litre, about 28 g/litre, about 33 g/litre, or about 38 g/litre.

In some examples, the serum-free cell culture medium as described hereinmay be protein-free. As used herein, the term “protein-free” refers to acell culture medium that is devoid of insulin or transferrin or growthfactors or any proteins purified from any organisms or any recombinantproteins. For clarity, hydrolysates or hydrolysed proteins may bepresent in protein-free cell culture medium. As serum contains proteins,all protein-free media are also serum-free.

In some examples, the serum-free cell culture medium as described hereinmay be a chemically defined medium. As used herein the term “chemicallydefined” refers to a growth medium suitable for the in vitro cellculture of cells in which all of the chemical components are identifiedand their exact concentrations known. A chemically defined medium mustalso be entirely free of any undefined components such as fetal bovineserum or serum from any other human or animal origin, or soyhydrolysate, yeast hydrolysate or any hydrolysates wherein the exactidentity and concentrations of components are not known. This means thata chemically defined media can contain recombinant versions of proteins,such as but not limited to, albumin and growth factors, usually derivedfrom rice or E. coli. As serum contains undefined components, allchemically defined media are also serum-free.

In some examples, the serum-free cell culture medium as described hereinmay be a protein-free chemically defined medium (PFCDM). As used hereinthe term “protein-free chemically defined” refers to a growth mediumsuitable for the in vitro cell culture of cells in which all of thechemical components are known, and is devoid of insulin or transferrinor growth factors or any proteins purified from any organisms or anyrecombinant proteins.

As illustrated throughout the Experimental Section below, the cellculture medium as described herein may further include, but is notlimited to, a basal cell culture medium selected from the groupconsisting of basal medium eagle (BME), Eagle's minimum essential medium(MEM or Eagle's MEM), Earle's balanced salt solution (EBSS), Dulbecco'smodified Eagle's medium (DMEM), HAM's F-10 medium, HAM's F-12 medium,DMEM-F12 medium, Roswell Park Memorial Institute 1640 (RPMI 1640 orRPMI), Leiboitz's medium (L-15-medium), combinations thereof or modifiedversions thereof. In some examples, the cell culture medium as describedherein may include Iscove's Modified Dulbecco's Medium (IMDM); IMDM withHEPES and L-Glutamine; IMDM with HEPES and without L-Glutamine; RPMI1640 with L-Glutamine; RPMI 1640 with HEPES, L-Glutamine and/orPenicillin-Streptomycin; Minimal Essential Medium-alpha (MEM-alpha);DMEM:F12 1:1 with L-Glutamine; DME/F12; Basal Medium Eagle with Earle'sBSS; GMEM (Glasgow's MEM); GMEM with L-glutamine; F-10; F-12; Ham's F-10with L-Glutamine; Ham's F-12 with L-Glutamine; L-15 (Leibovitz) (2×)without L-Glutamine or Phenol Red; L-15 (Leibovitz) without L-Glutamine;McCoy's 5A Modified Medium; Medium 199; MEM Eagle without L-Glutamine orPhenol Red (2×); MEM Eagle-Earle's BSS with L-glutamine; MEMEagle-Earle's BSS without L-Glutamine; MEM Eagle-Hanks BSS withoutL-Glutamine; NCTC-109 with L-Glutamine; Richter's CM Medium withL-Glutamine; and hydrolysate-containing media.

Furthermore, depending on the cells to be cultured, it is believed to beadvantageous if the cell culture medium as described herein may furthercomprise at least one additional ingredient that includes, but is notlimited to, at least one amino acid, at least one vitamin, at least oneinorganic salt, at least one trace element, adenine sulfate, ATP,deoxyribose, ethanolamine, ethanolamine.HCl, glutathione,N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),hypoxanthine, linoleic acid, lipoic acid, phenol red,phosphoethanolamine, putrescine, sodium pyruvate, cholesterol, dextransulphate, β-mercaptoethanol, methotrexate (MTX), methionine sulfoximine(MSX), thymidine, uracil, xanthine, combination thereof, and the like.

In some examples, the amino acid ingredient may include, but is notlimited to, one or more amino acids, such as, but not limited to,L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine,L-cysteine, L-glutamic acid, L-glutamine, glycine, L histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, Lproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, andthe like or combinations thereof.

The vitamin ingredient as described herein may include, but is notlimited to, one or more vitamins, such as, ascorbic acid, biotin,choline chloride, D-Ca++-pantothenate, folic acid, i-inositol,menadione, niacinamide, nicotinic acid, paraaminobenzoic acid (PABA),pyridoxal, pyridoxine, riboflavin, thiamine, vitamin A acetate, vitaminB12, vitamin D2, and the like or combinations thereof.

The inorganic salt ingredient as described herein may include, but isnot limited to one or more inorganic salts such as, CaCl₂, KCl, MgCl₂,MgSO₄, NaCl, NaHCO₃, Na₂HPO₄, NaH₂PO₄, KH₂PO₄, Ba(C₂H₃O₂)₂, KBr, CoCl₂,KI, MnCl₂, Cr(SO₄)₃, CuSO₄, NiSO₄, H₂SeO₃, NaVO₃, TiCl₄, GeO₂,(NH₄)₆Mo₇O₂₄, Na₂SiO₃, FeSO₄, NaF, AgNO₃, RbCl, SnCl₂, ZrOCl₂, CdSO₂,ZnSO₄, Fe(NO₃)₃, AlCl₃, ferric citrate chelate, and the like orcombinations thereof.

The trace element ingredient as described herein may include, but is notlimited to an ion of one or more trace elements such as barium, bromine,cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium,titanium, germanium, molybdenum, silicon, iron, fluorine, silver,rubidium, tin, zirconium, cadmium, zinc, aluminium, and the like orcombinations thereof.

It is also envisaged that the cell culture medium as described hereinmay be further combined with at least one, or at least two, or at leastall of ingredients such as a hydrolysate, or an enzymatic digest, oryeast cell extract.

In some examples, the cell culture medium as described herein may befurther combined with any one or more of a zwitterionic surfactant, anionic surfactant or a non-ionic surfactant. Suitable surfactants may beionic surfactants, which may either be an anionic surfactant or acationic surfactant. Anionic surfactants have a negative ionic group,either based on a permanent anion such as sulphate, sulfonate orphosphate, or on a pH-dependent anion such as carboxylate. Examples ofanionic surfactants includes, but is not limited to, alkyl sulfates,alkyl sulfonates, alkyl ether sulfates, alkyl phosphates, alkylphosphonates, docusates, sulfonate fluorosurfactants, alkyl benzenesulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, alkylcarboxylates, alkyl polyoxyethylene sulfates, carboxylatefluorosurfactants, ammonium lauryl sulfate, sodium dodecyl sulfate(SDS), dioctyl sodium sulfosuccinate, sodium deoxycholate, sodiumalginate, sodium-n-dodecylbenzenesulfonate, sodium lauryl sulfate,sodium lauryl ether sulfate (SLES), sodium myreth sulfate, dioctylsodium sulfosuccinate, phosphatidyl glycerol, potassium laurate,phosphatidyl inosine, phosphatidylinositol, perfluorooctanesulfonate(PFOS), perfluorobutanesulfonate, sodium stearate, triethanolaminestearate, diphosphatidylglycerol, phosphatidylserine, phosphatidic acidand their salts, sodium carboxymethylcellulose, cholic acid and otherbile acids (e.g., cholic acid, deoxycholic acid, glycocholic acid,taurocholic acid, glycodeoxycholic acid) and salts thereof (e.g., sodiumdeoxycholate), sodium lauroyl sarcosinate, perfluorononanoate, andperfluorooctanate (PFOA or PFO), and the like.

In some examples, the non-ionic surfactants may include, but is notlimited to, glyceryl esters, polyoxyethylene fatty alcohol ethers,polyoxyethylene sorbitan fatty acid esters (polysorbates),polyoxyethylene fatty acid esters, sorbitan esters, glycerolmonostearate, polyethylene glycols, polypropylene glycols, cetylalcohol, cetostearyl alcohol, stearyl alcohol, aryl alkyl polyetheralcohols, polyoxyethylene-polyoxypropylene copolymers (poloxamers),poloxamines, methylcellulose, hydroxymethyl cellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, noncrystalline cellulose,polysaccharides including starch and starch derivatives such ashydroxyethylstarch (HES), polyvinyl alcohol, polyvinylpyrrolidone, andthe like.

Cationic surfactants comprise a positive ionic group and pH-dependentcationic surfactants are based on primary, secondary or tertiary amines,whereas permanently charged cationic surfactants are based on quaternaryammonium cation. Examples of cationic surfactants may include, but isnot limited to, natural phospholipids, synthetic phospholipids,quaternary ammonium compounds, benzalkonium chloride, cetyltrimethylammonium bromide, chitosans, lauryl dimethyl benzyl ammonium chloride,acyl carnitine hydrochlorides, dimethyl dioctadecyl ammomium bromide(DDAB), dioleyoltrimethyl ammonium propane (DOTAP), dimyristoyltrimethyl ammonium propane (DMTAP), dimethyl amino ethane carbamoylcholesterol (DC-Chol), 1,2-diacylglycero-3-(O-alkyl) phosphocholine,O-alkylphosphatidylcholine, alkyl pyridinium halides, or long-chainalkyl amines such as, for example, n-octylamine, oleylamine, and thelike.

In some examples, the surfactant may be zwitterionic surfactants, whichare electrically neutral surfactants that posseses local positive andnegative charges within the same molecule. Examples of suitablezwitterionic surfactants include, but is not limited to, zwitterionicphospholipids such as phosphatidylcholine, phosphatidylethanolamine,diacyl-glycero-phosphoethanolamine (such asdimyristoyl-glycero-phosphoethanolamine (DMPE),dipalmitoyl-glycero-phosphoethanolamine (DPPE),distearoyl-glycero-phosphoethanolamine (DSPE),dioleolyl-glycero-phosphoethanolamine (DOPE)), and the like. In someexamples, the culture medium as described herein may include mixtures ofphospholipids that include anionic and zwitterionic phospholipids. Suchmixtures include, but are not limited to lysophospholipids, egg orsoybean phospholipid, or any combination thereof. The phospholipid,whether anionic, zwitterionic or a mixture of phospholipids, may besalted or desalted, hydrogenated or partially hydrogenated or naturalsemi-synthetic or synthetic.

In some examples, the surfactant may include, but is not limited to,fatty alcohols; polyoxyethylene glycol octylphenol ethers; andpolyoxyethylene glycol sorbitan alkyl esters. In other examples, thesurfactant may be a non-ionic surfactant. In some examples, thesurfactant may include, but is not limited to, polysorbate 80 (PS80),polysorbate 20 (PS20), and poloxamer 188 (P188).

To prevent undesired growth, it may be advantageous for the cell culturemedium as described herein to comprise agent that prevents undesiredgrowth of contaminants. Thus, in some examples, the cell culture mediumas described herein may further comprise an antibiotic agent. Theantibiotic agent may include, but is not limited to, hygromycin B,puromycin, blasticidin, bleomycin sulfate, geneticin (G418), zeocin,amphotericin B, ampicillin, penicillin, chloramphenicol, gentamycin,kanamycin, neomycin, streptomycin, tetracycline, polymyxin B,actinomycin D, amikacin, bacitracin, carbenicillin, ceftazidime,coumermycin A1, D-cycloserine, cyclohexamide, dihydrostreptomycinsesquisulfate, kasugamycin, mycophenolic acid, nalidixic acid,nourseothricin sulfate, oxytetraclycline, paromomycin sulfate,phleomycin, mitomycin C ribostamycin, rifampicin, rifamycin,spectinomycin, tazobactam, thiostrepton, ticarcillin, and combinationsthereof.

It would be understood to the person skilled in the art that eachingredient as described herein will be present in an amount that issuitable for the cultivation of the desired cell in vitro. Thus, in oneexample, each ingredient of the culture medium as described herein maybe present in an amount which supports the cultivation of a cell invitro. As would be understood, it is envisaged that increasing theconcentrations of other nutrients in the batch culture medium mayfurther improve protein (such as recombinant protein) productivity.

To assist in the transportation, and/or storage of the cell culturemedium as described herein, the cell culture medium as described hereinmay be provided as a cell culture medium concentrate. Thus, in someexamples, the cell culture medium as described herein may be a 1× to a100× medium formulation, or a 1×, or a 2×, or a 5×, or a 10×, or a 50×,or 100× medium formulation. The culture medium as described herein maybe provided in powdered form or liquid form.

In some example, the cell culture medium may include, but is not limitedto, Dulbecco's minimum essential medium (DMEM), F-12 basal medium(DMEM-F12), L-glutamine, a non-ionic surfactant, geneticin and maltose,or a modified version thereof.

As illustrated in the Experimental Section below, the cell culturemedium may further include glucose. Therefore, in some examples, thecell culture medium may include, but is not limited to, Dulbecco'sminimum essential medium (DMEM), F-12 basal medium (DMEM-F12),L-glutamine, a non-ionic surfactant, geneticin, maltose and glucose, ora modified version thereof.

Depending on the purpose of the cell culture, the cell to be cultured inthe culture medium as described herein may include, but is not limitedto, a vertebrate cell, an arthropod cell, an annelid cell, a molluscscell, a sponge cell, a jellyfish cell, an insect cell, an avian cell, amammalian cell, a fish cell, and the like.

When the cell to be cultured is an insect cell, the insect cell may bederived from Spodoptera spp. or Trichoplusa spp.

In some examples, the cell to be cultured in the culture medium asdescribed herein may be a mammalian cell. The mammalian cell mayinclude, but is not limited to, a human cell, a murine cell, a rat cell,a hamster cell, a rabbit cell, a dog cell, a monkey cell, a hybridomacell, a CHO cell, a CHO-K1 cell, a CHO-DG44 cell, a CHO-S cell, aCHO-DXB11 cell, a CHO-GS cell, a SH87 cell, a BHK cell, a COS cell, aVERO cell, a HeLa cell, a 293 cell, a PER-C6 cell, a K562 cell, a MOLT-4cell, an M1 cell, an NS-1 cell, a COS-7 cell, an MDBK cell, an MDCKcell, an MRC-5 cell, a WI-38 cell, a WEHI cell, an SP2/0 cell, a CAPcell, a AGELHN cell, or a derivative thereof. In some examples, themammalian cell may be a CHO-K1 cell, or a CHO-DG44 cell, or a protein(such as recombinant protein) producing derivative thereof, such asSH87.

Advantageously, as demonstrated in the Experimental Section below, theculture medium as described herein may be used to culture any cells,without the need of any modifications to enable the metabolism ofalternative carbohydrate source (such as maltose). Thus, in someexamples, the cell that may be used for the cell culture medium asdescribed herein may be able to metabolize the sole carbohydrate sourceor carbohydrate sources without requiring prior adaptation to saidsource/sources. As used herein, the term “adaptation” refers toadjusting or changing the physiology of an organism in order to be moresuited to an environment. This adjustment or change can be performed,for example by genetic modification of said organism, but can also takeplace spontaneously through natural selection and environmentalselective pressures.

In another aspect, there is provided a method of growing and/orculturing a cell, wherein the method comprises growing and/or culturinga cell in the serum-free cell culture medium as described herein. Insome examples, the method may comprise growing and/or culturing a cellin the serum-free cell culture medium that may comprise maltose as solecarbohydrate source or may comprise maltose and at least one, or atleast two, or at least three, or at least four, or more saccharides ascarbohydrate sources. In some examples, the methods may comprise growingand/or culturing a cell as described herein, wherein the culture mediummay comprise maltose and glucose as sole carbohydrate sources or theculture medium may comprise Dulbecco's minimum essential medium (DMEM),F-12 basal medium (DMEM-F12), L-glutamine, a non-ionic surfactant,geneticin, and maltose, or a modified version thereof. As shown in theExperimental Section below, the method of growing and/or culturing thecell as described herein improves protein production. Thus, in someexamples, the method as described herein improves protein yield.

The method of growing and/or culturing a cell as described herein maygrow cells using methods of cell culturing known in the other. In someexamples, the cells as described herein may be grown and/or cultured incultures including, but not limited to, adherent cultures, suspensioncultures, T-flask cultures, spinner flask cultures, shake flaskcultures, spin-tube cultures, microbioreactor cultures, bioreactorcultures, in batch cultures, in fed-batch cultures, in continuouscultures, in perfusion cultures, and the like. In some examples, thegrowing and/or culturing of the cell may be performed under conditionssuitable for supporting growth and/or culture of the cell.

As shown in the Experimental Section, the methods as disclosed hereincan increase protein yield. Thus, in another aspect, there is provided amethod of increasing protein yield. The method comprises growing and/orculturing a cell in the serum-free cell culture medium as describedherein. In some examples, the method may comprise growing and/orculturing a cell in the serum-free cell culture medium that may comprisemaltose as sole carbohydrate source or may comprise maltose and at leastone, or at least two, or at least three, or at least four, or moresaccharides as carbohydrate sources. In some examples, the methods maycomprise growing and/or culturing a cell as described herein, whereinthe culture medium may comprise maltose and glucose as sole carbohydratesources or the culture medium may comprise Dulbecco's minimum essentialmedium (DMEM), F-12 basal medium (DMEM-F12), L-glutamine, a non-ionicsurfactant, geneticin, and maltose, or a modified version thereof.

In some examples, the increased protein yield may be increased by about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100%. In some examples, the increase ofprotein yield may be an increase in the production of antibody, whereinthe increase in antibody titer may be by about 2% to about 300%, orabout 2%, or about 4%, or about 6%, or about 8%, or about 10%, or about12%, or about 14%, or about 16%, or about 18%, or about 20%, or about22%, or about 24%, or about 26%, or about 28%, or about 30%, or about32%, or about 34%, or about 36%, or about 38%, or about 40%, or about43%, or about 45%, or about 48%, or about 50%, or about 55%, or about60%, or about 65%, or about 70%, or about 75%, or about 80%, or about90%, or about 100%, or about 125%, or about 150%, or about 175%, orabout 200%, or about 230%, or about 250%, or about 300%.

In some examples, the method as described herein may increase cellspecific productivity by about 2% to about 300%, or about 2%, or about4%, or about 6%, or about 8%, or about 10%, or about 12%, or about 14%,or about 16%, or about 18%, or about 20%, or about 22%, or about 24%, orabout 26%, or about 28%, or about 30%, or about 32%, or about 34%, orabout 36%, or about 38%, or about 40%, or about 43%, or about 45%, orabout 48%, or about 50%, or about 55%, or about 60%, or about 65%, orabout 70%, or about 75%, or about 80%, or about 90%, or about 100%, orabout 125%, or about 150%, or about 175%, or about 200%, or about 230%,or about 250%, or about 300%.

In some examples, the cell culture media and/or methods as describedherein may increase maximum viable cell density/number by about 2% toabout 300%, or about 2%, or about 4%, or about 6%, or about 8%, or about10%, or about 12%, or about 14%, or about 16%, or about 18%, or about20%, or about 22%, or about 24%, or about 26%, or about 28%, or about30%, or about 32%, or about 34%, or about 36%, or about 38%, or about40%, or about 43%, or about 45%, or about 48%, or about 50%, or about55%, or about 60%, or about 65%, or about 70%, or about 75%, or about80%, or about 90%, or about 100%, or about 125%, or about 150%, or about175%, or about 200%, or about 230%, or about 250%, or about 300%, asmeasured over a course of, for example, 14 days.

As described in FIG. 8 and Table 3, the cell culture medium as describedherein and/or the methods as described herein may be used to fine-tuneglycosylation profiles of the protein product (e.g. recombinantglycoprotein product). In one aspect, there is provided a method ofmodulating glycosylation profile of a protein, wherein said methodcomprises culturing a cell expressing said protein in a cell culturemedia as described herein, to thereby produce a cell that expresses theprotein with a modulated glycosylation profile. In some example, themodulation of glycosylation profile of the protein may be the modulationof glycosylation such as, but is not limited to, antennae, fucosylation,a mannosylation, a sialylation, or combination thereof, and the like.

As used herein, the term “modulation” refers to an increase or decreaseof a specific type of glycosylation of the protein as compared to acontrol. As used herein, the “control” medium may be a cell culturemedium that is the same as the culture medium as described herein withthe exception of the control does not contain maltose. That is, thecontrol may be an equivalent cell culture medium of the cell culturemedium as described herein with the absence of maltose. The onlydifference between the control cell culture medium and the cell culturemedium as described herein is the presence of disaccharide (such asmaltose) in the cell culture medium as described herein.

In some examples, the modulated glycosylated profile includes, but isnot limited to, a decreased level of fucosylated glycans, a decreasedlevel of diantennary glycans, an increased level of mannosylatedglycans, an increased level of mono-antennary glycans, a decreasedsialylated glycans, or combination thereof. Therefore, in one example,there is provided a method of producing a protein having at least one,at least two, at least three, or all of the modulated glycosylationincluding, but is not limited to, a decreased level of fucosylatedglycans, decreased level of diantennary glycans, increased mannosylatedglycans, increased mono-antennary glycans, and decrease sialylatedglycans. In some examples, as illustrated in Table 3, the protein may bean antibody, or an immunoglobulin, or fragments thereof.

In some examples, the modulation of the fucosylation level may be adecrease in the fucosylation level in the protein. In some examples, thedecrease in the level of fucosylated glycans may be a decrease of about0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65%.

In some examples, the modulation of the mannosylated glycan level may bean increase in the mannosylation level of the protein. In some examples,the mannosylation level may be an increase of about 0.1%, 1%, 1.2%,1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45% or 50%.

In some examples, the modulation of the sialylation level may be adecrease in the sialylation level in the protein. In some examples, thedecrease in the level of sialylated glycans may be a decrease of about0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65%.

In some examples, the modulation of the mono-antennary glycan level maybe an increase in the mono-antennary glycan level of the protein. Insome examples, the mono-antennary glycan level may be an increase ofabout 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%,4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

In some examples, the modulation of the diantennary glycan level may bea decrease in the diantennary glycan level in the protein. In someexamples, the decrease in the level of diantennary glycan may be adecrease of about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%,4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60% or 65%.

As used herein, the term “antennae” refers to the addition of GlcNAcsequences to glycan core in hybrid and complex glycans. Thus, a“mono-antennary glycans” refers to one GlcNAc branch linked to the coreglycan, a “diantennary glycans” refers to two GlcNAc branches linked tothe core glycan, and the like.

In one example, there is provided a method of producing compositionscomprising an antibody, or antigen binding fragment thereof, with amodulated glycosylation profile. The methods may include culturing ahost cell expressing the antibody, or antigen binding fragment thereof,in cell culture media as described herein, thereby producing thecomposition comprising the antibody, or antigen binding fragmentthereof, with at least one (at least two, or all) selected from thegroup consisting of a 0.1-50% decrease in the level of fucosylatedglycans, a 0.1-50% decrease in the level of sialylated glycans, and a0.1-50% increase in the level of mannosylated glycans as compared to acontrol. In some examples, the control is a composition comprising anantibody, or antigen binding fragment thereof, produced by culturing ahost cell expressing the antibody, or antigen binding fragment thereof,in cell culture media which is not cultured in the culture media asdescribed herein. In one example, the antibody is an anti-Her2 antibody,or an antigen binding fragment thereof. In one example, the culturemedia comprises glucose and/or maltose.

As shown in FIG. 8A, maltose supplementation may also reduce thesialylation level in the antibody expressed. It is known that reductionof sialylation level of an antibody may improve the antibody-dependentcellular cytotoxicity (ADCC) of therapeutic antibodies. As such, in someexamples, the present disclosure also provide methods of producingcompositions comprising an antibody, or antigen binding fragmentthereof, with a modulated glycosylation profile by culturing a host cellexpressing the antibody, or antigen binding fragment thereof, in cellculture media as described herein, thereby producing the compositioncomprising the antibody, or antigen binding fragment thereof, with a0.1-30% increase in antibody-dependent cellular cytotoxicity (ADCC)response as compared to a control, wherein the control is a compositioncomprising an antibody, or antigen binding fragment thereof, produced byculturing a host cell expressing the antibody, or antigen bindingfragment thereof, in cell culture media which is not a cell culturemedium as described herein. In one example, the antibody is an anti-Her2antibody, or an antigen binding fragment thereof. In one example, theculture media comprises glucose and/or maltose.

The cell culture mediums as described herein and the methods asdescribed herein may be used for culturing and/or growing and/orincreasing the protein yield of cells such as a vertebrate cell, anarthropod cell, an annelid cell, a molluscs cell, a sponge cell, ajellyfish cell, an insect cell, an avian cell, a mammalian cell, a fishcell, and the like. In some examples, the cell culture mediums asdescribed herein and the methods as described herein may be used forculturing and/or growing and/or increasing the protein yield ofeukaryotic cells or tissues including animal cells, human cells, insectcells, plant cells, avian cells, fish cells, mammalian cells and thelike.

In some examples, the mammalian cell may include, but is not limited to,a human cell, a murine cell, a rat cell, a hamster cell, a rabbit cell,a dog cell, a monkey cell, a hybridoma cell, a CHO cell, a CHO-K1 cell,a CHO-DG44 cell, a CHO-S cell, a CHO-DXB11 cell, a CHO-GS cell, a SH87cell, a BHK cell, a COS cell, a VERO cell, a HeLa cell, a 293 cell, aPER-C6 cell, a K562 cell, a MOLT-4 cell, an M1 cell, an NS-1 cell, aCOS-7 cell, an MDBK cell, an MDCK cell, an MRC-5 cell, a WI-38 cell, aWEHI cell, an SP2/0 cell, a CAP cell, a AGE1.HN cell, or a derivativethereof.

The culture medium as described herein increased the immunoglobulinproduction of cells. Thus, in some examples, the cell to be cultured inthe cell culture and methods as described herein may be anantibody-producing cell. In some examples, the mammalian cell may be aCHO-K1 cell, or a CHO-DG44 cell, or a recombinant protein producingderivative thereof, such as SH87.

For convenience, the culture medium may be provided as a kit. Thus, inone aspect, there is provided a kit comprising the components of thecell culture medium as described herein and/or the components forperforming the methods as described herein. In some examples, the kitmay further comprise a cell. For example, the cell may be the cell to becultured. The cell may include, but is not limited to, a vertebratecell, an arthropod cell, an annelid cell, a molluscs cell, a spongecell, a jellyfish cell, an insect cell, an avian cell, a mammalian cell,a fish cell, and the like. In some examples, the mammalian cell mayinclude, but is not limited to, a human cell, a murine cell, a rat cell,a hamster cell, a rabbit cell, a dog cell, a monkey cell, a hybridomacell, a CHO cell, a CHO-K1 cell, a CHO-DG44 cell, a CHO-S cell, aCHO-DXB11 cell, a CHO-GS cell, a SH87 cell, a BHK cell, a COS cell, aVERO cell, a HeLa cell, a 293 cell, a PER-C6 cell, a K562 cell, a MOLT-4cell, an M1 cell, an NS-1 cell, a COS-7 cell, an MDBK cell, an MDCKcell, an MRC-5 cell, a WI-38 cell, a WEHI cell, an SP2/0 cell, a CAPcell, a AGELHN cell, and the like, or a derivative thereof.

As exemplified in the Experimental Section, the mammalian cell may be aCHO-K1 cell, or a CHO-DG44 cell, or a protein (such as recombinantprotein) producing derivative thereof, such as SH87.

In some examples, the cell to be cultured in the culture medium asdescribed herein and/or methods as described herein may be a cell thatproduces protein such as, but is not limited to, recombinant protein,antibodies, and the like. As illustrated in the Experimental Sectionbelow, the protein may be monoclonal antibodies.

The disclosure illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims and non-limitingexamples. In addition, where features or aspects of the invention aredescribed in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group.

Experimental Section

Materials and Methods

Cell Lines and Cell Cultivation

CHO-K1 cell line was previously adapted to suspension culture in aserum-free protein-free medium, HyQ PF-CHO MPS (Hyclone, Logan, Utah)supplemented with 2 g/l sodium bicarbonate (Sigma-Aldrich, St. Louis,Mo.), 3.6 g/l D-(+)-Glucose (Sigma-Aldrich), 6 mM L-Glutamine(Sigma-Aldrich) and 0.1% Pluronic® F-68 (Life Technologies, Carlsbad,Calif.). SH87, a suspension CHO-K1 cell line that is producing ananti-Her2 monoclonal antibody (Ho et al., 2012), was previously adaptedto a DMEM/F12-based protein free chemically defined medium (PFCDM)supplemented with 6 g/l D-(+)-Glucose (Sigma-Aldrich), 8 mM L-Glutamine(Sigma-Aldrich), 0.1% Pluronic F-68 (Life Technologies), and 600 μg/mlG418 disulfate salt (Sigma-Aldrich). Both CHO-K1 and SH87 cells wereroutinely passaged every 3 to 4 days.

Unless otherwise specified, cell cultures in this disclosure wereperformed in single-use Erlenmeyer flasks (Corning, Acton, Mass.)incubated in a humidified incubator (Climo-Shaker ISF-1-W, Kuhner,Switzerland) at 37° C., 8% CO² and a rotation speed of 110 rpm.

Analysis of Cell Culture Samples

Viable cell density and culture viability were determined by Trypan bluedye exclusion method using Vi-Cell XR Cell Viability Analyzer (BeckmanCoulter, Brea, Calif.) according to manufacturer's instructions. Forbiochemical and other cell culture parameter analyses, 1 ml of culturesample was centrifuged at 8000 g for 10 minutes to obtain clarifiedsupernatant.

Concentrations of ammonium, glutamine, glucose and lactate were analyzedby the BioProfile 100 Plus (Nova Biomedical, Waltham, Mass.). Osmolalitywas measured using a vapor pressure osmometer (Vapro 5520, Wescor,Logan, Utah), according to manufacturer's instructions. Maltoseconcentration was quantified using Maltose Colorimetric/FluorometricAssay Kit (Biovision, Milpitas, Calif.), according to manufacturer'sinstructions. Monoclonal IgG antibody titer was determined bynephelometry using IMMAGE 800 (Beckman Coulter), according tomanufacturer's instructions.

Adaptation of CHO Cell Lines into Culture Media with DifferentDisaccharides

HyQ PF-CHO disaccharide media were prepared by replacing the 3.6 g/lglucose normally added to the medium, with the same mass concentrationsof maltose, sucrose, lactose or trehalose during media preparation.CHO-K1 cells were then seeded into these disaccharide media at cellseeding densities of 0.3×10⁶ cells/ml and 1.0×10⁶ cells/ml, and passagedevery 3 to 4 days. At each passage during the adaptation process, viablecell density and culture viability before passage and after inoculationinto fresh media were determined.

Shake Flask Batch Cultures Sampling and Characterization

For growth curve comparison of CHO-K1 cells in maltose and glucosemedia, CHO-K1 cells adapted to the maltose medium and non-adapted CHO-K1cells were seeded into HyQ PFCHO disaccharide medium with 3.6 g/lmaltose and the normal HyQ PF-CHO medium with 3.6 g/l glucose,respectively. For the evaluation of maltose utilization usingnon-adapted SH87, cells were seeded into PFCDM with 4, 6, 14 or 24 g/lglucose, or 4 g/l glucose supplemented with 0.5, 1, 2, 3, 10 or 20 g/lmaltose. Cells were cultivated in single-use Erlenmeyer flasks(Corning), with a cell seeding density of 0.3×10⁶ cells/ml induplicates. Cell culture samples were collected and analyzed dailythroughout the duration of the growth profile experiment until cultureviabilities fell below 50%. When necessary, culture supernatant sampleswere stored at −20° C. for further analysis.

Quantification of Intracellular Maltose

Samples for intracellular maltose quantification were prepared by firstquenching 10⁷ cells with ice cold 150 mM NaCl (Merck, ACS Reagent Grade)solution. After the cells were pelleted by centrifugation, thesupernatant was removed by aspiration and 10 μl of 0.4 mM 13C-maltose(UL-¹³C₁₂ maltose monohydrate, Omicron Biochemicals, South Bend, Ind.)was added as a reference standard. A two-phase liquid extractionprotocol, involving the use of methanol (Fisher Scientific, Optimagrade), chloroform (Merck) and tricine (Merck) solution (40:35:25 v/v)(Selvarasu et al., 2012) was utilized to extract intracellular maltose.The extracts were stored at −80° C., dried under vacuum at a temperatureof 4° C. (CentriVap, Labconco, US) and reconstituted in a water-methanolmixture (95:5 v/v) before analysis via liquid chromatography-massspectrometry (LC-MS) (Acquity UPLC-Xevo TQ-S MS, Waters, Milford,Mass.). The separation was performed using a C18 reverse phase column(Waters, HSS T3 column, 2.1 mm×50 mm, 1.8 μm particle size), with thefollowing solvents—A: water with 0.1% formic acid (Sigma-Aldrich, 98%),B: Methanol, at a flow rate of 0.4 ml/min. Quantification ofintracellular maltose was carried out via multiple reaction monitoringexperiments, in which the integrated peak areas for maltose and13C-maltose in each sample were obtained. The actual concentration ofmaltose in each sample was quantified by direct comparison of therelative integrated peak area of maltose to that of the 13C-maltosereference standard. The lower limit of detection for maltose wasobserved to be 7.5 ng per injection. Intracellular maltose concentrationwas calculated based on maltose quantities within detection limit,number of cells used and average cell diameter obtained from Vi-Cell XRCell Viability Analyzer. The samples were analyzed on LC-MS intriplicate and the integrated peak areas obtained for each sample wereobserved to have relative standard deviations of less than 15%.

Bioreactor Fed-Batch Culture Sampling and Characterization

For each culture condition to be tested, SH87 was scaled up in PFCDM andinoculated into duplicate two (2) liter glass bioreactors (Sartorius,Germany) at a viable cell density of 3×10⁵ cells/ml. Culturetemperature, pH, dissolved oxygen and stir rate were maintained at 37°C., 7.1, 50% and 120 rpm respectively. The culture set points forglucose and glutamine were 0.5 g/l and 0.5 mM respectively, andpredictive feeding was used to maintain these concentration levels bythe addition of a concentrated DMEM-based protein-free feed and a 150g/l glucose solution. Cell culture samples were collected and analyzeddaily throughout the duration of the growth profile experiment untilculture viabilities fell below 50%. When necessary, culture supernatantsamples were stored at −20° C. for further analysis.

Antibody Glycosylation Analysis

Antibody glycosylation analysis was performed according to previouslypublished protocol with modifications (Chan et al., 2015). Briefly,Protein-A-purified IgG samples were first desalted using a PD 10 column(GE Healthcare, Pittsburgh, Pa.) following manufacturer's protocol.Then, glycans were released and labeled with RapiFluor MS (RFMS) (Lauberet al., 2015) according to manufacturer's protocol (Waters Corporation,Milford, Mass.). After labeling, excess RFMS was removed by passing thelabeling mixture through a MiniTrap G-10 desalting column (GEHealthcare) and the purified RFMS-labeled glycans were then dried undervacuum. The samples were reconstituted in 200 μl reconstitution buffercontaining 42.8 μl of water, 50 μl of dimethylformamide and 107.2 μl ofacetonitrile, and analyzed by the UNIFI Biopharmaceutical platform(Waters Corporation, Milford, Mass.). Raw retention time of eachchromatographic peak obtained was converted to a glucose unit (GU) byfitting into a calibration curve established by a RFMS-labeled dextranladder (Waters Corporation). The observed GU value and the associatedmass of each chromatographic peak were then used to search against anexperimental database for N-glycans embedded in the UNIFIBiopharmaceutical platform, which contains information on expected GUvalues and masses of more than 300 N-glycan species. A structure is thenassigned to each chromatographic peak based on two orthogonalcriteria: 1) the observed GU value matches the expected GU value within0.2 GU deviation, and 2) the observed mass matches the expected mass ofthe glycan within 5 ppm mass error. Additionally, knowledge of CHOglycosylation features was applied as biological filter to removeirrelevant candidate structures, such as glycans with bisecting GlcNAcand α2,6-linked sialic acid.

Calculations

Specific growth rate (μ) was determined by plotting ln(VCD) vs taccording to Equation 1, where VCD is the viable cell density, VCD₀ isthe initial viable cell density and t is the culture time.

VCD=VCD₀ e ^(μt)

ln(VCD/VCD₀)=μt  Equation 1

The cumulative integrated viable cell density (IVCD) was calculated bytrapezium rule according to Equation 2.

IVCD_(t)=IVCD_(t-1)+0.5×(VCD_(t)+VCD_(t-1))×Δt  Equation 2

Specific IgG productivity (qp) between culture times t1 and t2 wasdetermined by plotting IgG titer (P) vs IVCD according to Equation 3.

q _(p)=(P _(t2) −P _(t1))/IVCD

P _(t2) =q _(p)×IVCD+P _(t1)  Equation 3

Specific substrate consumption rate (qs) between culture times t1 and t2was determined by plotting substrate concentration (S) vs IVCD accordingto Equation 4.

−qs=(S _(t2) −S _(t1))/IVCD

S _(t2) =−q _(s)×IVCD+S _(t1)  Equation 4

Specific substrate consumption rate (qs) was fitted into a Monod modelaccording to Equation 5, where qs_max is the maximum specific substrateconsumption rate, S is the substrate concentration, and Ks is theaffinity constant.

q _(s)=(q _(s) _(_) _(max) ×S)/(K _(s) +S)  Equation 5

Results and Discussion

Evaluation of Disaccharides to Support Growth of CHO-K1 Cells

To evaluate the use of disaccharides to support the growth of mammaliancells, a Chinese Hamster Ovary (CHO) cell line, CHO-K1, was cultivatedusing a seeding cell density of 0.3×10⁶ cells/ml, with 3.6 g/l ofmaltose, sucrose, lactose, trehalose or glucose as energy source in aserum-free protein-free cell culture medium HyQ PF-CHO. Osmolality ofthese culture media were determined to be between 308 and 324 mOsm/kg,well within the range for optimal mammalian cell culture. The viablecell densities and culture viabilities of these cultures at thebeginning and end of each passage over a period of 74 days are shown inFIG. 1. While the cells in glucose containing medium grew to highculture viabilities and viable cell densities at each passage, thoseparameters for cells in disaccharide media decreased and remainedstagnant respectively. However, culture viabilities and viable celldensities of the maltose culture started to pick up on Day 14.Proliferation of the cells in maltose culture then maintained over theperiod of the time studied, while those in the other disaccharides wereterminated on Day 31 due to the lack of cell growth and depressedculture viabilities. The same experiment repeated using a higher seedingcell density of 1.0×10⁶ cells/ml gave similar results (data not shown).Hence, the present disclosure demonstrated that CHO-K1 cells canproliferate in serum-free protein-free culture medium utilizing maltose,but not sucrose, lactose or trehalose, as sugar source.

To validate the observation, the growth profile of the adapted CHO-K1cell line in the maltose protein-free medium was then compared to theCHO-K1 cells cultivated in the glucose protein-free medium, usingsimilar initial cell density and sugar concentration of 0.3×10⁶ cells/mland 3.6 g/l respectively (FIG. 2). The cells cultivated with maltose asenergy source grew to a much lower maximum viable cell density of1.4×10⁶ cells/ml on Day 7 compared to that with glucose which reached5.9×10⁶ cells/ml on Day 6 (FIG. 2A). As the initial sugar concentrationwas the same, the inventors of the present disclosure postulated thatthis difference may be due to the depletion of other nutrients such asglutamine, since glutamine was depleted at similar rates till completelyutilized on Days 4 and 5 for the glucose and maltose culturesrespectively (FIG. 2D). Using viable cell density data till Day 4, thedoubling time of the maltose culture was 53.7 h compared to 22.3 h forthe glucose culture, showing that the rate of energy metabolism may belimiting the cell growth rate in the maltose culture. Interestingly, themaltose culture maintained at high culture viabilities greater than 80%over a longer period of time, compared to cells cultivated in glucose(FIG. 2A). This may be due in part to the lower maximum cell densitythat the maltose culture reached, which in turn may have resulted inhigher amounts of un-metabolized nutrients and a lower accumulation oftoxic metabolites at later time points of the culture. Another reasonmay be that the cells in the maltose culture had a lower metabolism dueto the limiting energy uptake rate, and this resulted in less cellularstress and slower cell death.

Examining critical biochemicals in the culture medium, glucose was notdetectable in the maltose culture (FIG. 2B), confirming the lack ofglucose in the medium from the onset of the culture. As glucoseconcentration maintained at an undetectable level throughout theculture, this shows that maltose was not hydrolyzed into glucose in theculture medium by secreted maltases, but was consumed by the cells. Thisconsumption may have occurred via one of the three (3) possiblemechanisms: (1) maltose may be transported into the cells prior tohydrolysis by intracellular maltase such as acid alpha glucosidase(GAA), or (2) maltose may be hydrolysed by plasma membrane boundmaltase, such as intestinal maltase-glucoamylase

(MGAM) and sucrase-isomaltase (SI), and immediately taken up by theglucose transporters that are also found on the plasma membrane. Theseadditional steps for energy metabolism may be rate limiting to result inthe slow growth rate of the maltose culture, as discussed above. Thelack of lactate production in the maltose culture (FIG. 2C) supportsthis hypothesis as it shows that the cells in the maltose cultureutilized the more energy efficient Krebs cycle, rather than ending theglycolysis pathway with lactate production, as embodied by the lactategeneration in the glucose culture (FIG. 2C).

Examining glutamine consumption, while this was slower in the maltoseculture compared to the glucose culture (FIG. 2D), it was likely due tothe slower growth of the maltose culture. This is verified by acomparison of the specific glutamine consumption of the glucose and themaltose cultures: By plotting the glutamine profiles of the culturesagainst the IVCD, it was observed that the specific glutamineconsumption, given by the slope of the curve, is similar between the twocultures (FIG. 2F). This demonstrates that glutamine consumption rate ofeach cell was unaffected by the use of maltose. Given the slower growthrate and lower maximum cell density of the maltose culture, the similarspecific glutamine consumption rate shows that more glutamine in themaltose culture was being channelled into energy metabolism instead ofcellular replication, when compared to the glucose culture. Thishypothesis is supported by the ammonium production profiles: while thespecific ammonium production rate was initially similar for bothcultures, that for the glucose culture slowed considerably from Day 2(FIG. 2G) to attain a final ammonium concentration of 0.14 g/l (FIG.2E), whereas the specific ammonium production rate of the maltoseculture continued at a similar rate till Day 5 to attain a higher finalammonium concentration of 0.20 g/l. The slower specific ammoniumproduction rate of the glucose culture from Day 2 can be attributed tothe higher cell growth rate (FIG. 2A) and a similar ammonium productionrate, showing that more amino acids were utilized for cell replicationcompared to the maltose culture. The difference in the final ammoniumconcentrations further supports this, as it shows that more amino acidsare subjected to de-amidation to supplement the energy consumption ofthe cells in the maltose culture when compared to the glucose culture,since both cultures have the same starting concentration of glutamineand other amino acids. It was also noted that the time at which ammoniumproduction plateaued, at Day 4 and Day 6 for the glucose and maltosecultures respectively, corresponded with the times of glutaminedepletion for the respective cultures, showing the ammonia accumulationmay be partly due to spontaneous glutamine deamidation. When thesemaltose-adapted CHO-K1 cells were further adapted a DMEM/F12-basedprotein free chemically defined medium (PFCDM) and tested with increasedconcentrations of maltose from 10 g/l to 40 g/l, the cells proliferatedfaster, showing that maltose can be used as a glucose-replacement in theabsence of hydrolysates in routine cultivation of mammalian cells (FIG.10).

Taken together, this data confirms that the cells do proliferate in themaltose medium in the absence of serum or protein supplement, althoughthe cells grow at a slower rate possibly due to rate limiting energymetabolism. Specific consumption of glutamine of the maltose cultureremained similar when compared to glucose culture, showing thatessential nutrients may be depleted to limit the maximum viable celldensity achievable by the maltose culture. The similar specificglutamine consumption rate and increased ammonium production in themaltose culture further shows that more amino acids may be deamidated inthe maltose culture to supplement energy metabolism, when compared tothe glucose culture.

The survival and proliferation of CHO-K1 cells in maltose containingprotein-free medium is surprising because there is no known mammalianmaltose transporter and mammalian cells are typically known to be unableto metabolize disaccharides, unless secreted or transmembrane maltasesare expressed, for example in intestinal cells. Although CHO cells havebeen shown to survive using polysaccharides as energy sources, theseexperiments were performed in serum containing media (Morgan and Faik,1981), and it was demonstrated that enzymes in serum breaks down thesepolysaccharides for the cells to metabolize (Scannell and Morgan, 1982).Hence, the present disclosure surprisingly demonstrates the serum-freeprotein-free mammalian cell culture as disclosed herein, when comprisesa disaccharide, can provide sufficient energy source for cell growth.

Application of Maltose to Sustain Culture Viability Upon GlucoseDepletion

Since CHO-K1 cells can utilize maltose as energy source and maintainhigh cell culture viability for extended periods in protein-free culturemedium containing maltose in the absence of glucose (FIG. 2), theinventors of the present disclosure proceeded to evaluate whethermaltose can be utilized to sustain the culture viability of non-adaptedCHO-K1 production cell lines upon glucose depletion. The cells werecultivated in a medium containing both glucose and maltose, to examinewhether the cells can grow using glucose in the medium for normal cellgrowth, followed by a switch to maltose metabolism to maintain cultureviability and protein production. This will potentially extend theapplication of maltose supplementation to non-adapted cell lines, andalso be a simple method to extend cell culture viability for higherrecombinant protein productivity in batch cultures.

For this evaluation, a CHO-K1 cell line (SH87) that is producing ananti-Her2 monoclonal antibody (Ho et al., 2012) was used. Batch shakeflask cultures of these cells in a DMEM/F12-based protein freechemically defined medium (PFCDM) with 4 g/l glucose, 6 g/l glucose, or4 g/l glucose supplemented with different concentrations of maltose weremonitored till culture viabilities dropped below 50% (FIG. 3). 4 g/lglucose was chosen as the base glucose concentration because this willallow for a premature but controlled cell growth limitation that ismainly due to glucose depletion in the batch cultures for this PFCDM.This controlled growth limitation is observed in FIG. 3A where the 4 g/lglucose culture reached maximum viable cell density (VCD) of 8.5×10⁶cells/ml on Day 5, one day earlier than the 6 g/l glucose culture, whichreached maximum VCD of 11.9×10⁶ cells/ml on Day 6. This corresponded tothe glucose depletion for the two cultures, which occurred one day priorto reaching the maximum VCDs (FIG. 3B). As such, by supplementing the 4g/l glucose culture medium with maltose, the effects of additionalmaltose on this non-adapted CHO-K1 production cell line can bedetermined without the added complications of the depletion of othernutrients.

From FIG. 3A, it was observed that all cultures with maltosesupplementation have a higher maximum VCD and longer culture viabilitycompared to the 4 g/l glucose only culture, even though the glucose inthese culture media were depleted at the same time (FIG. 3B).

Recombinant protein production was also maintained in the maltosesupplemented cultures to result in a higher IgG titer compared to the 4g/l glucose only culture (FIG. 3E). When compared to the 6 g/l glucoseonly culture, the maximum VCD and IgG titers of the maltose supplementedculture were lower (FIGS. 3A and G), with the exception of the IgG titerof the 3 g/l maltose supplemented culture, which matched that of the 6g/l glucose culture. This demonstrates that while maltose can supportcell growth when glucose was depleted before the 6 g/l glucose culture(FIG. 3B), metabolism is slower in the maltose supplemented cultures toresult in slower growth rates. This was further supported by theobservation that the growth improvement due to maltose wasconcentration-dependent (FIG. 3A), showing that the rate of maltosemetabolism may be limiting at these concentrations. This correspondedwith the ammonium production profiles too (FIG. 3G), which showed higherammonium production for the 4 g/l glucose cultures with 0, 0.5 and 1 g/lmaltose supplement, despite similar glutamine consumption profiles whencompared to the cultures with 6 g/l glucose or that with 3 g/l maltosesupplement (FIG. 3F). This shows that more amino acids are subjected todeamidation to supplement the energy consumption in the cultures with noor low concentrations of maltose. On the other hand, the 3 g/l maltosesupplemented culture has a lower ammonia production that was similar tothat of the 6 g/l glucose culture (FIG. 3G). This shows that the rate ofmaltose metabolism was sufficient for the cells' reduced energyrequirements at this maltose concentration during the later phase of thebatch culture.

When lactate profiles are examined, it was noted that lactateconsumption occurred with the depletion of glucose for all cultures(FIG. 3C). In contrast to glucose-only cultures where lactateconsumption was partial, all lactate was consumed at Day 6 formaltose-supplemented cultures. It was postulated that the completelactate consumption may be facilitated by maltose metabolism which keptthe culture viable and metabolizing in a low glucose environment for alonger time to utilize the lactate. This property of maltosesupplemented cultures may be useful in biopharmaceutical productionbecause lactate accumulation commonly causes cell toxicity in fed-batchbioreactor production processes (Hassell et al., 1991; Lao and Toth,1997). Interestingly, lactate production was observed again from Day 6onwards for the culture supplemented with 3 g/l maltose, supporting thehypothesis that the maltose metabolism rate was sufficient for the cellsto survive on glycolysis at this maltose concentration.

When the culture supernatant maltose concentrations were analyzed, itwas observed that maltose was indeed consumed by the cells, and thatmost consumption occurred when glucose was depleted (FIG. 3D). Thisindicates that these cells preferentially utilized glucose for growth,and when the glucose was depleted, the culture switched to maltosemetabolism which helped to maintain culture viability (FIG. 3A).Interestingly, maltose was depleted at about the same day even thoughthe initial maltose concentrations vary by up to a 6 fold difference.This can be partially attributed to the difference in VCD in the variousmaltose-supplemented culture, but it also shows that maltose metabolismmay be concentration-dependent at these maltose concentrations,resulting in higher maltose utilization at higher maltoseconcentrations.

To determine whether maltose could be internalized by the cells, SH87cell samples from both the 4 g/l glucose-only culture and the 3 g/lmaltose-supplemented culture were obtained for LC-MS analysis (FIG. 3H).While intracellular maltose was detected 1 day after seeding into themaltose-supplemented culture medium, it was found to be absent (belowdetection limits) in cells cultivated in glucose-only medium orimmediately after seeding into the maltose-supplemented medium. Thisconfirmed the presence of an intracellular maltose pool in the culturessupplemented with 3 g/l maltose, and demonstrates that maltose did enterthese cells, despite the lack of known transport mechanism, addingfurther evidence that maltose is indeed utilized by the cells in themaltose-supplemented cultures. In addition, further hints regardingmaltose transport were noted from the intracellular maltoseconcentrations, which were maintained at levels approximately 100 timeslower than that in the culture medium over the 5 days monitored: Thisshows that the transport of maltose may be actively regulated to preventits accumulation inside the cells despite the concentration gradient,since the cells only switched to maltose metabolism upon glucosedepletion (FIG. 3D).

To further validate that the maltose was indeed utilized by the cellsand not hydrolyzed in the culture media, cell-free conditioned media(CM) from Days 2, 4, 6 and 8 were obtained from a 3 g/l maltosesupplemented culture as well as a 4 g/l glucose culture. 3 g/l maltosewas spiked into the CM from the glucose culture, and both sets of CMwere monitored over 3 days for changes in glucose and maltoseconcentrations at 37° C. (FIG. 4). The growth profiles of the cultureswere similar to those in FIG. 3A, with the maltose supplemented culturehaving a higher viable cell density and extended culture viability atDays 6 and 8 when compared to the glucose culture (FIG. 4A). Glucose andmaltose profiles for the maltose supplemented culture were also similar,with glucose depleting on Day 6 and maltose being depleted between Days4 and 8 (FIG. 4 B). On the other hand, the CM harvested from the maltosesupplemented culture maintained the same glucose and maltoseconcentrations at which they were harvested, despite being incubated atthe same temperature over 3 days (FIG. 4 B). Similarly, the CM from theglucose culture that were spiked with 3 g/l maltose maintained atconsistent glucose and maltose concentrations despite the low cultureviabilities on Days 6 and 8 (FIG. 4 C). This demonstrates that thepresence of cells was necessary for the utilization of the maltose, andthat maltose hydrolysis was not occurring spontaneously in theconditioned culture media, even when culture viabilities were low. Sincemaltose can be hydrolyzed by pancreatic and salivary α-amylases whichare secreted proteins, intestinal maltase-glucoamylase andsucrose-isomaltase which are transmembrane proteins, and lysosomalα-glucosidase and neutral α-glucosidase C which are intracellularproteins, this data confirms that the secreted maltases were notinvolved in the maltose metabolism in these CHO cells.

As the culture medium as described herein does not contain serum,hydrolysates nor proteins, the present disclosure precluded the roles ofundefined media components in these observations, and conclusivelyproved that CHO-K1, a mammalian cell line, can utilize maltose forgrowth and recombinant protein production. Furthermore, this data showsthat the cells do not need prior adaptation to utilize maltose and tosustain culture viability in a biphasic manner.

Comparison of Batch Cultures with High Glucose and MaltoseConcentrations

As it was observed that maltose metabolism may be limiting atconcentrations of 3 g/l or less, the culture profiles of SH87 in mediasupplemented with 4 g/l glucose and 10 g/l or 20 g/l maltose wascompared to cultures of the same cells in media containing 14 g/l and 24g/l glucose (FIG. 5). The 24 g/l glucose culture grew slower, reached alower maximum VCD of 10.6×106 cells/ml, and reached a low viability ofless than 50% faster than the other 3 cultures (FIG. 5A). The slowergrowth of the 24 g/l glucose culture can be attributed to the higherosmolality (360 mOsm/kg) of the culture medium (FIG. 5F), while theculture media of the other 3 cultures had similar osmolality between 318and 331 mOsm/kg that were within the range for optimal mammalian cellculture. This confirms a limitation of high glucose loading in batchculture medium because glucose contributes to osmolality significantlyand can affect cell growth at high concentrations. In contrast, the samemass concentration of maltose contributed to less increase in osmolalityand thus had no negative effect on cell growth.

Comparing the growth profiles of the 10 g/l maltose, 20 g/l maltose and14 g/l glucose cultures (FIG. 5A), they were similar even after glucoseconcentration reached its minimal on Day 5 for the maltose supplementedcultures (FIG. 5B). This shows that the rate of maltose metabolism isnot limiting cell growth at these concentrations, in contrast to theslower growth observed at maltose concentrations lower than 3 g/l afterglucose depletion (FIGS. 3A and B). The hypothesis that the maltosemetabolism was higher at these concentrations is supported byobservations of glucose accumulation in the 10 g/l and 20 g/l maltosesupplemented cultures (FIG. 5B), a phenomenon not observed previously(FIG. 3B). This shows that maltose was hydrolyzed faster than the cells'metabolic requirements during the corresponding culture times: Glucoseaccumulation was observed in the 20 g/l maltose supplemented cultureafter Day 5, when the cells were still growing at a similar rate as the14 g/l glucose culture, demonstrating that maltose metabolism was notlimiting cell growth at this maltose concentration. At the lower maltoseconcentration of 10 g/l, glucose accumulation was observed only afterDay 8, when the cells are entering death phase. With the cells growingsimilarly to the 14 g/l glucose culture, this illustrates that maltosemetabolism rate was just ample to support cell growth at 10 g/l maltosewithout excess glucose accumulating in the culture medium.

Also worth noting here is that the lactate concentrations of the glucoseonly cultures accumulated throughout the culture duration, in contrastto the maltose supplemented cultures which consumed most of the lactatefrom Days 5 to 8 (FIG. 5C). While a second lactate production phase wasobserved after Days 9 and 10 for the 10 g/l and 20 g/l maltosesupplemented cultures respectively, the lactate concentrations in thesecultures reached significantly lower levels as compared to the glucosecultures. This verifies another limitation of glucose loading in batchculture medium: because lactate accumulation can result in cell toxicity(Hassell et al., 1991; Lao and Toth, 1997), the lack of its consumptionwith high glucose loading can limit the growth of the cell culture. Inthis case, this lactate toxicity may have contributed to the fasterdeath phase observed for the 24 g/l glucose culture.

Examining the maltose profiles of the maltose supplemented cultures,most maltose were consumed after glucose depletion on Day 5 (FIG. 5D),similar to previous data (FIG. 3D), and maltose was not depleted forboth maltose supplemented cultures till the end of the run on Day 14.Similarly, glucose was not depleted for both glucose cultures (FIG. 5B).While sugars were present in excess, other nutrients may be limiting thegrowth of these cells, for example, glutamine was depleted at Day 6, atsimilar rates for all 4 cultures (FIG. 5E). These other limitations mayhave affected growth similarly to result in the similar growth profilesof the maltose supplemented cultures and that of the 14 g/l glucoseculture.

With the similar growth profiles, it was interesting to note that the 20g/l maltose supplemented culture resulted in a maximum IgG titer of 298mg/1, 15% higher than that obtained from the 14 g/l glucose culture and10 g/l maltose supplemented cultures which gave maximum titers of 259mg/l and 263 mg/l respectively (FIG. 5G). To examine this, the specificIgG productivities of the cultures were determined according to Equation3 (Table 1 and FIG. 11). It was noted that the specific IgGproductivities for all 4 cultures decreased after Day 7, when glutamineconcentrations reached their minimum, showing that glutamine may belimiting IgG productivity from Day 7. Regardless of glutaminelimitation, it was observed that specific IgG productivities were 21 to29% higher in the culture with more glucose or with more maltose,showing that higher sugar concentrations can improve specific IgGproductivities. When the specific IgG productivities of maltosesupplemented cultures was compared with their corresponding glucoseculture having the same total sugar concentration, the maltosesupplemented cultures had 13 to 17% lower specific IgG productivities upto Day 6, though these became 25 to 26% higher than specific IgGproductivities of their corresponding glucose culture from Day 7. Hence,the presence of maltose had negatively impacted specific IgGproductivities prior to glutamine depletion, but contributed to improvedspecific IgG productivities after glutamine depletion. As such, thehigher sugar content of the 20 g/l maltose supplemented culture resultedin 7 to 53% higher specific IgG productivities when compared to the 14g/l glucose culture and the 10 g/l maltose supplemented culturerespectively, and this had led to the higher maximum IgG titers of the20 g/l maltose supplemented culture, despite the similar growthprofiles.

TABLE 1 Specific IgG productivities of SH87 cultivated in protein-freechemically defined medium (PFCDM) with high concentrations of glucoseand maltose. SH87 cell routinely maintained in glucose-only PFCDM wassub-cultivated into PFCDM with 14 g/l glucose, 24 g/l glucose, 4 g/lglucose + 10 g/l maltose, or 4 g/l glucose + 20 g/l maltose. IgG titerswere then plotted against IVCD to obtain the specific IgG productivityas the slope of the graphs, according to Equation 3. Culture media sugarSpecific IgG productivity Specific IgG productivity content up to Day 6(pg/cell/day) from Day 7 (pg/cell/day) 14 g/l glucose 7.74 0.96 24 g/lglucose 10.0 1.17 4 g/l glucose + 6.73 1.21 10 g/l maltose 4 g/lglucose + 8.29 1.47 20 g/l maltose

Characterization of Maltose Metabolism Kinetics

To characterize the maltose metabolism in the maltose supplementedcultures, the specific maltose consumption rates were obtained fromplots of maltose concentrations against cumulative integral viable celldensities (IVCD) according to Equation 4. These plots gave straighttrendlines with R2 values between 0.853 and 0.999 when 3 to 5 datapoints after glucose depletion were used (FIG. 12), showing that thespecific maltose consumption rates were fairly constant when maltose wasmetabolized. The slopes of these trendlines gave the average specificmaltose consumption rates of these cultures and are tabulated in FIG.6A. The specific maltose consumption rates increased with increasinginitial maltose concentrations, verifying that maltose metabolism wasindeed concentration dependent at these maltose concentrations.Additionally, it was observed that the magnitude of increase in specificconsumption decreased at higher initial maltose concentrations, showingthat there may be a maxima in this relationship. As such, the data wasfitted to a Monod model according to Equation 5 by non-linearregression. The data fitted to the model (FIG. 6B) to obtain a maximumspecific maltose consumption rate (qs_max) of 0.257 ng/cell/day and anaffinity constant (Ks) of 7.03 g/l. While it was noted that a modellingthe effect of maltose concentration on specific growth rate may bemeaningful, it was not practicable with this data since cell growth weremostly minimal after glucose depletion. Comparing with publishedspecific monosaccharide consumption rates using 3.6 g/l ofmonosaccharides (Altamirano et al., 2000), the maximum specific maltoseconsumption rate determined here is similar to the specific consumptionrates of fructose (0.21 ng/cell/day) and galactose (0.21 ng/cell/day)while being lower than the measured specific consumption rates ofglucose (0.76 ng/cell/day) and mannose (0.88 ng/cell/day). This showsthat maltose can be metabolized at rates similar to fructose andgalactose while maltose metabolism will be slower than that with glucoseand mannose. While the present inventors noted that these parameters canbe more accurately determined in continuous cultures, it may bepractically challenging at low maltose concentrations since growth rateswill be low. Hence, the current data gave a surprising demonstrationthat mammalian cell may have maltose metabolism kinetics.

Application of Maltose in Fed-Batch Cultures

Since fed-batch culture is a popular mode for the manufacturing ofmonoclonal antibodies, the use of maltose in fed-batch cultures wasevaluated. In contrast to batch cultures where initial glucoseconcentration is limited by the detrimental effect on cell growth due toconsequent increase in osmolality beyond a certain limit, fed-batchcultures do not have this limitation since glucose can be fedcontinually into the cultures. As such, it was evaluated whether maltosecan be used to supplement glucose in fed-batch cultures to drive thecells towards a slower but more efficient metabolism: Using a commonglucose concentration setpoint of 0.5 g/l, SH87 in media supplementedwith 4 g/l glucose and 20 g/l maltose was fed daily with 50% of itscalculated glucose requirement, while cultures of the same cells inmedia containing 4 g/l glucose was fed daily with 100% of its calculatedglucose requirement. Duplicate bioreactor cultures were performed foreach condition and the growth, biochemical and IgG titer profiles ofthese cultures were plotted in FIG. 7.

In contrast to the lactate consumption observed in the batch cultures(FIGS. 3 and 5), there was no lactate consumption in the fed-batchcultures, even in the maltose-supplemented cultures with a 50% glucosefeed (FIG. 7C). It was postulated that this may be due to the consistentnutrient feeding in the fed-batch cultures, whereas glucose, glutamineor other nutrients were likely depleted in the batch cultures to resultin the observed lactate consumption. Nonetheless, IgG titers offed-batch cultures (FIG. 7G) were more than 5 fold that obtained fromthe batch cultures (FIG. 5G), demonstrating that the feeding strategywas successful in improving IgG production as expected of fed-batchcultures.

Comparing between the fed-batch cultures, the viable cell densities,culture viabilities and lactate profiles of the maltose-supplementedcultures were similar to Glucose-only #2 culture, while Glucose-only #1culture had a higher maximum viable cell density, a faster decrease inculture viability and a faster lactate accumulation (FIGS. 7A and C).The faster increase in osmolality in glucose-only culture #1 (FIG. 7F)is likely due to pH correction as a result of the faster lactateaccumulation. These illustrate possible variability of these parametersin the SH87 fed-batch cultures. On the other hand, glucose and glutamineprofiles of maltose supplemented and glucose-only cultures were similar.In addition, IgG titer was fairly consistent between the replicatecultures despite the variability in viable cell density, lactate andosmolality observed in the glucose-only duplicate cultures.

Comparing the glucose profiles, it was interesting to note that despitebeing fed only at 50% of its calculated glucose requirement, themaltose-supplemented cultures maintained similar culture glucoseconcentrations as the glucose-only fed-batch cultures. Nonetheless,because of this reduced glucose feeding, specific glucose consumption ofthe maltose-supplemented cultures was 0.115±0.007 ng/cell/day which is45% that of the glucose-only cultures at 0.254±0.013 ng/cell/day (Table2). These show that the maltose-supplemented cultures were most probablyusing a secondary energy source in addition to glucose to achievecomparable growth and culture viability profiles as Glucose-only #2culture. This secondary energy source is likely to be maltose, becausespecific glutamine consumption rates were similar between themaltose-supplemented and glucose-only cultures (Table 2), and maltoseconsumption was observed in the maltose-supplemented cultures from Day7, one day after glucose feeding was initiated in the maltosesupplemented fed-batch cultures (FIG. 7D).

TABLE 2 Maximum IgG titers, specific production and consumption rates ofSH87 cultivated in protein-free chemically defined medium (PFCDM) withand without maltose supplement in fed-batch bioreactor cultures. SH87cell routinely maintained in glucose-only PFCDM was sub-cultivated induplicate fed-batch bioreactor cultures (denoted by #1 and #2) asdescribed in FIG. 7. Specific growth rates were determined according toEquation 1. Cumulative amounts of biochemicals produced by the cellswere plotted against cumulative Integral Viable Cell (IVC) number toobtain the specific productivities as the slope of the graphs. MaltoseAverage ± Standard supplement #1 #2 deviation Maximum IgG titer + 19201745 1833 ± 124  (mg/l) − 1435 1530 1483 ± 67  Maximum IgG titer + 31.226.3 28.8 ± 3.5  (mg/l) − 15.6 21.5 18.6 ± 4.1  Exponential specific +0.49 0.501 0.496 ± 0.008 growth rate (day-1) − 0.696 0.607 0.652 ± 0.063Specific glucose + 0.11 0.119 0.115 ± 0.007 consumption (ng/cell/day) −0.263 0.245 0.254 ± 0.013 Specific glutamine + 0.036 0.03 0.033 ± 0.004consumption (ng/cell/day) − 0.029 0.027 0.028 ± 0.001 Specific lactate +0.106 0.114 0.110 ± 0.005 production (ng/cell/day) − 0.117 0.077 0.097 ±0.028

With the maltose supplemented metabolism, it was observed that therewere 23% and 55% improvements in maximum IgG titers and specific IgGproductivities at 1.833±0.124 g/l and 28.8±3.5 pcd, from the 1.483±0.067g/l and 18.6±4.1 pcd observed in glucose-only fed-batch culturesrespectively (Table 2). When compared to Glucose-only #2 culture with amore comparable growth profile which has maximum IgG titers and specificIgG productivities at 1.53 g/l and 21.5 pcd respectively, theimprovements in maximum IgG titers and specific IgG productivities were20% and 34% respectively.

One possible mechanism for the observed improvement in IgG productionmay be the higher initial osmolality of the maltose-supplementedcultures due to the additional 20 g/l maltose: This may have resulted inthe 22% to 40% lower specific growth rates of the maltose-supplementedcultures compared to the glucose-only cultures (Table 2), to possiblyallow slower and more productive maltose-supplemented cultures.Nonetheless, when maltose-supplemented batch culture was compared toglucose-only batch culture having similar initial osmolality (FIG. 5G),an improvement in IgG titer was surprisingly similarly observed. Thisshows that the improvement in maximum IgG titers observed in the maltosesupplemented fed-batch cultures is not only due to osmolality effect:Without wishing to be bound by theory, it is postulated that a loweravailability of glucose, enabled by the presence of maltose, may haveresulted in a more efficient cell metabolism in the maltose-supplementedculture, to also contribute in the observed higher maximum IgG titers.

To verify whether this lower availability of glucose can have the sameeffect in the absence of maltose, SH87 glucose-only fed-batch culturesfed was compared with 100% or 50% of their calculated glucoserequirements in a separate experiment (FIG. 13). While similar maximumviable cell densities were reached in both conditions before glucosefeeding was initiated, the culture viabilities of the glucose-onlycultures with 50% calculated glucose feed dropped below 50% 4 daysearlier than the cultures with 100% calculated glucose feed. Maximum IgGtiters of the 50% fed cultures were also only 46.4% that of the 100% fedcultures. These confirmed that the presence of maltose enabled the useof a lower glucose feed to result in the observed higher maximum IgGtiters in FIG. 7G.

As glycosylation is a critical attribute of therapeutic IgG products,purified IgG from glucose only and maltose supplemented cultures fromDays 10 and 15 were subjected to glycosylation profiling (FIG. 8). Therepresentative fluorescence chromatograms of Day 10 samples (FIG. 8A)showed that the glycosylation profiles of IgG from both glucose-only andmaltose supplemented cultures were similar, showing that maltosesupplementation did not grossly affect the glycan profiles of themonoclonal antibodies produced. When the relative abundance of glycanstructures of the IgG from maltose supplemented cultures were comparedto that from glucose-only cultures (FIG. 8B), it was noted that therewere marginally less fucosylated, sialylated, G1F, G2F and biantennaryglycans, and more GOF and monoantennary glycans. Most of thesedifferences were also observed when glycans from Day 15 samples werecompared to that from Day 10 samples, showing that maltosesupplementation affected glycan profiles in a way that is mostly similarto a later harvest. The exceptions to the similarity are in the relativeabundances of the sialylated and high-mannose glycans: while the laterDay 15 harvests gave higher levels of high-mannose glycans and similarlevels of sialylated glycans compared to Day 10 harvests, maltosesupplementation gave lower levels of sialylated glycans and similarlevels of high-mannose glycans compared to glucose-only cultures. Thisdata shows that maltose supplementation can also be used as a means tofine-tune monoclonal antibody glycosylation profile, especially inmarginally reducing the sialylation level, which is known to improveantibody-dependent cellular cytotoxicity (ADCC) of therapeuticantibodies. Additionally, this may also be important in the field ofbiosimilar manufacturing, where matching of the biosimilar glycanprofile to the innovator drug is an important criterion.

Effect of Maltose Supplementation in Both Basal and Feed Media inFed-Batch Cultures

Additionally, the present disclosure investigated whether maltosesupplementation in the feed medium can have further effect on fed-batchcultures with maltose-supplemented base medium. Using a common glucoseconcentration setpoint of 0.5 g/l, SH87 in media supplemented with 4 g/lglucose and 20 g/l maltose was fed daily with 50% of its calculatedglucose requirement, using a sugar feed consisting of only glucose, orof glucose and maltose in a 1:1 ratio.

Duplicate bioreactor cultures were performed for each condition and thegrowth, biochemical and IgG titer profiles of these cultures wereplotted in FIG. 9. It was observed that additional maltosesupplementation in the feed medium had no additional effect on the cellgrowth, viability, glutamine, osmolality and IgG titer profiles. This islikely because the initial maltose supplementation was not exhausted inthe maltose-supplemented culture with glucose-only feed. It would beexpected that the addition of maltose in the feed medium may have aneffect if the initial maltose was exhausted in longer fed-batch runs,which can be achieved if temperature and/or pH shift were implemented.It was noted that the additional maltose supplementation in the feedmedium increased the glucose concentration and decreased the lactateconcentration marginally when compared to the culture with glucoseonlyfeed. This may be advantageous in cultures whereby lactate build-up iscausing the culture to crash.

Samples from these cultures were similarly subjected to glycosylationanalysis. The differences in glycosylation profiles were shown in Table3.

TABLE 3 Effect of maltose supplementation in feed medium onglycosylation profile. Differences in glycosylation profiles ofanti-Her2 antibody produced from SH87 fed-batch bioreactor cultures inglucose only, or glucose + maltose protein-free chemically definedmedium (PFCDM) base medium with glucose-only feed (Glucose + Maltose) orglucose + maltose feed (Glucose + Maltose_BF). Anti-Her2 monoclonalantibodies were purified from samples from Day 10 and Day 15 of the SH87fed-batch cultures and subjected to glycosylation profiling. Thedifferences in relative abundance of glycan structures were calculatedusing data from two biological replicates. Differences in the effects ofmaltose supplemented feed compared to maltose culture with glucose-onlyfeed on the relative abundance of glycan structures were in bold. 1(Glucose + 2 3 Maltose_BF)- (Glucose + Glucose + (Glucose + Maltose)-Maltose_BF)- 4 Maltose) (Glucose only) (Glucose only) Day 15-Day 10Average SD Average SD Average SD Average SD Fucosylated −0.81 0.44 −0.390.33 −1.19 0.41 −1.21 0.39 Glycans Sialylated 0.14 0.40 −0.73 0.71 −0.590.52 −0.19 0.55 Glycans High-mannose 0.39 0.38 0.03 0.27 0.42 0.36 0.730.34 Glycans Antennary 1 0.83 0.55 1.50 0.32 2.33 0.49 1.30 0.45Antennary 2 −0.83 0.52 −1.41 0.50 −2.24 0.65 −1.99 0.56 G0F −0.99 1.172.58 1.09 1.59 1.58 4.75 1.28 G1F −0.14 0.87 −2.36 1.21 −2.50 1.52 −5.761.20 G2F −0.14 0.40 −1.09 0.43 −1.23 0.50 −1.09 0.44

Although maltose supplementation in the feed medium has no effect on thetiter of the antibody produced, it gave marginally less fucosylated anddiantennary glycans and more high mannose and mono-antennary glycanswhen compared to the maltose culture with glucose-only feed. Comparingcolumn 3 to column 4 in Table 3, it was noted that having maltose in thefeed pushed the glycosylation profile of the maltose supplementedculture to be more similar to that of a later harvest. Thesedemonstrates that maltose supplementation in the feed medium can be usedto complement the maltose supplemented basal medium to finetuneglycosylation profiles of the recombinant glycoprotein product.

Evaluation of Disaccharides to Support Growth of CHO-DG44 Cells

To determine whether the same approach can be applied on other CHO celllines, the use of disaccharides to support the growth of CHO-DG44 cellswas evaluated. These cells were cultivated with 10 g/l of maltose,sucrose, lactose, trehalose or glucose as energy source in a serum-freeprotein-free cell culture medium HyQ PF-CHO. The viable cell densitiesand culture viabilities of these cultures at the beginning and end ofeach passage over a period of 22 days are shown in FIG. 1B. While thecells in glucose and maltose containing medium grew to high cultureviabilities and viable cell densities at each passage, those parametersfor cells in other disaccharide media decreased and remained stagnant.Hence, it was demonstrated that CHO-DG44 cells can also proliferate inserum-free protein-free culture medium utilizing maltose, but notsucrose, lactose or trehalose, as sugar source. It would be expectedthat CHO-DG44 production cells will have similar improvements in growthand product titer with maltose supplementation, as the presentdisclosure has demonstrated with CHO-K1 cells.

In this disclosure, it was surprisingly demonstrated that CHO-K1 cellscan utilize maltose for growth in the absence of serum or proteinsupplement, although the cells grew at a slower rate. In addition, whenculture media with both glucose and maltose were used, prior celladaptation was not necessary for the utilization of maltose, whichfollowed glucose depletion in a biphasic manner. The utilization ofmaltose was dependent on the presence of cells in the culture, asmaltose was internalized by the cells and maltose hydrolysis did notoccur spontaneously in the conditioned culture media. The practicalapplication of maltose supplementation to increase the carbohydratecontent of cell culture medium was also shown, since an increasedglucose concentration is limited by the corresponding increase inosmolality. The utilization of maltose in batch cell culture has anadded advantage of promoting lactate consumption, which will otherwiseaccumulate and become toxic to the cells. These factors contributed to a15% improvement in the recombinant monoclonal antibody titer from batchcultures. The specific maltose consumption rates obtained from batchcultures were fitted in a Monod model to obtain a maximum specificmaltose consumption rate (qs_max) of 0.257 ng/cell/day and an affinityconstant (Ks) of 7.03 g/l.

It was further demonstrated that maltose supplementation can also beapplied to fed-batch bioreactor cultures to result in 23% and 55%improvements in maximum monoclonal antibody titers and specificmonoclonal antibody productivities respectively, when compared toglucose-only fed-batch cultures. This shows that maltose supplementationcan be applied as a simple bioreactor process modification to improvemonoclonal antibody yields in current monoclonal antibody manufacturingfed-batch processes. Glycosylation profiling of the antibodies producedfrom the fed-batch cultures shows that maltose supplementationmarginally affected glycan profile similar to a later harvest, with anadditional effect of slightly reducing sialylation levels without aconcomitant increase in high-mannose glycans. This shows that maltosesupplementation can also be applied to marginally affect monoclonalantibody glycosylation profile, which is important in affecting the ADCCof the antibody therapeutic and in the physical matching of biosimilarglycan profile to that of the innovator drug.

In addition to the practical implications of maltose supplementation onbiopharmaceutical production, the ability of CHO-K1 cells to utilizemaltose in protein-free medium is surprising in itself because there isno known mammalian maltose transporter and mammalian cells are typicallyknown to be unable to metabolize disaccharides. Hence, the presentdisclosure provides for surprising evidence of a serum-free protein-freemammalian cell culture using a disaccharide as the energy source. Alsodisclosed is an estimate of maltose metabolism kinetics in mammaliancells.

Furthermore, the present disclosure has demonstrated that maltosesupplementation in the feed medium can have marginal effects on theglycosylation profiles of the glycoprotein product, and that CHODG44cells can also utilize maltose as a carbohydrate source.

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1. A serum-free cell culture medium comprising maltose as solecarbohydrate source or a serum-free cell culture medium comprisingmaltose and at least one additional saccharide as carbohydrate sources.2. (canceled)
 3. The serum-free cell culture medium of claim 1, whereinthe saccharide is a monosaccharide.
 4. (canceled)
 5. The serum-free cellculture medium of claim 1, comprising maltose and glucose as solecarbohydrate sources.
 6. The serum-free cell culture medium of claim 1,wherein the saccharide is a polysaccharide or a disaccharide.
 7. Theserum-free cell culture medium of claim 6, wherein the polysaccharide isa glucan. 8-9. (canceled)
 10. The serum-free cell culture medium ofclaim 1, wherein the serum-free cell culture medium is protein-free. 11.The serum-free cell culture medium of claim 1, wherein the serum-freecell culture medium is a chemically defined medium.
 12. The serum-freecell culture medium of claim 1, wherein the serum-free cell culturemedium is a protein-free chemically defined medium (PFCDM).
 13. Theserum-free cell culture medium according to claim 1, wherein the cellculture medium further comprises a hydrolysate, an enzymatic digest oryeast cell extract.
 14. The serum-free cell culture medium according toclaim 1, wherein the cell culture medium is further comprises an ionicsurfactant or a non-ionic surfactant.
 15. The serum-free cell culturemedium according to claim 1, wherein the cell culture medium furthercomprises an antibiotic agent.
 16. The serum-free cell culture medium ofclaim 1, wherein each ingredient is present in an amount which supportsthe cultivation of a cell in vitro.
 17. The serum-free cell culturemedium of claim 1, wherein said at least one saccharide is present at aconcentration of between 0.5 g/litre to 40 g/litre.
 18. The serum-freecell culture medium of claim 1, wherein maltose is present at aconcentration of between 0.5 g/litre to 40 g/litre.
 19. The serum-freecell culture medium of claim 1, wherein the serum-free cell culturemedium comprises F-12 basal medium (DIEM-F12), L-glutamine, a non-ionicsurfactant, geneticin and maltose, or a modified version thereof. 20.(canceled)
 21. The serum-free cell culture medium of claim 1, whereinthe serum-free cell culture medium is in powdered or liquid form. 22.The serum-free cell culture medium of claim 1, wherein the cell isselected from the group consisting of a vertebrate cell, an arthropodcell, an annelid cell, a molluscs cell, a sponge cell, a jellyfish cell,an insect cell, an avian cell, a mammalian cell and a fish cell.
 23. Theserum-free cell culture medium of claim 22, wherein the cell is able tometabolize the sole carbohydrate source or carbohydrate sources withoutrequiring prior adaptation to said source/sources. 24.-25. (canceled)26. A method of growing and/or culturing a cell or a method ofincreasing protein yield, wherein the method comprises growing and/orculturing a cell in a serum-free cell culture medium comprising maltoseas sole carbohydrate source or a serum-free cell culture mediumcomprising maltose and at least one additional saccharide ascarbohydrate sources. 27.-30. (canceled)
 31. A method of modulatingglycosylation profile of a protein, wherein the method comprisesculturing a cell expressing the protein in a serum-free cell culturemedium to thereby produce a cell that expresses the protein with themodulated glycosylation profile, wherein the serum-free culturecomprises maltose as sole carbohydrate source or a serum-free cellculture medium comprising maltose and at least one additional saccharideas carbohydrate sources. 32.-35. (canceled)